Identification of Surface-Associated Antigens for Tumor Diagnosis and Therapy

ABSTRACT

The invention relates to the identification of gene products that are the result of tumor-associated expression and the nucleic acids encoding the same. The invention also relates to the therapy and diagnosis of diseases wherein these gene products are the result of an aberrant tumor-associated expression. The invention also relates to the proteins, polypeptides and peptides that are the result of tumor-associated expression and to the nucleic acids encoding the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 15/009,061, filed onJan. 28, 2016, which is a continuation of U.S. Ser. No. 12/851,980,filed on Aug. 6, 2010, now U.S. Pat. No. 9,255,131, which is a divisionof U.S. application Ser. No. 11/596,106, filed on Jun. 26, 2007, nowU.S. Pat. No. 7,785,801, which is the National Stage of PCT/EP05/005104,filed on May 11, 2005; each of which is incorporated herein by referencein its entirety.

INCORPORATION OF SEQUENCE LISTING

Biological sequence information for this application is included in anASCII text file having the file name “VOS-204-1-SEQ.txt” created on Mar.31, 2014, and having a file size of 395,736 bytes, which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath. More recent therapeutic concepts aim at incorporating thepatient's immune system into the overall therapeutic concept by usingrecombinant tumor vaccines and other specific measures such as antibodytherapy. A prerequisite for the success of such a strategy is therecognition of tumor-specific or tumor-associated antigens or epitopesby the patient's immune system whose effector functions are to beinterventionally enhanced. Tumor cells biologically differ substantiallyfrom their nonmalignant cells of origin. These differences are due togenetic alterations acquired during tumor development and result, interalia, also in the formation of qualitatively or quantitatively alteredmolecular structures in the cancer cells. Tumor-associated structures ofthis kind which are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens.

The specific recognition of tumor-associated antigens involves cellularand humoral mechanisms which are two functionally interconnected units:CD4⁺ and CD8⁺ T lymphocytes recognize the processed antigens presentedon the molecules of the MHC (major histocompatibility complex) classesII and I, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens.

The potential clinical-therapeutical importance of tumor-associatedantigens results from the fact that the recognition of antigens onneoplastic cells by the immune system leads to the initiation ofcytotoxic effector mechanisms and, in the presence of T helper cells,can cause elimination of the cancer cells (Pardoll, Nat. Med. 4:525-31,1998). Accordingly, a central aim of tumor immunology is to molecularlydefine these structures. The molecular nature of these antigens has beenenigmatic for a long time. Only after development of appropriate cloningtechniques has it been possible to screen cDNA expression libraries oftumors systematically for tumor-associated antigens by analyzing thetarget structures of cytotoxic T lymphocytes (CTL) (van der Bruggen etal., Science 254:1643-7, 1991) or by using circulating autoantibodies(Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To thisend, cDNA expression libraries were prepared from fresh tumor tissue andrecombinantly expressed as proteins in suitable systems. Immunoeffectorsisolated from patients, namely CTL clones with tumor-specific lysispatterns, or circulating autoantibodies were utilized for cloning therespective antigens.

In recent years a multiplicity of antigens have been defined in variousneoplasias by these approaches. The class of cancer/testis antigens(CTA) is of great interest here. CTA and genes encoding them(cancer/testis genes or CTG) are defined by their characteristicexpression pattern [Tureci et al, Mol Med Today. 3:342-9, 1997]. Theyare not found in normal tissues, except testis and germ cells, but areexpressed in a number of human malignomas, not tumor type-specificallybut with different frequency in tumor entities of very different origins(Chen & Old, Cancer J. Sci. Am. 5:16-7, 1999). Antibodies against CTAare not found in healthy individuals but in tumor patients. This classof antigens, in particular owing to its tissue distribution, isparticularly valuable for immunotherapeutic projects and is tested incurrent clinical patient studies (Marchand et al., Int. J. Cancer80:219-30, 1999; Knuth et al., Cancer Chemother. Pharmacol. 46:p46-51,2000).

However, the probes utilized for antigen identification in the classicalmethods illustrated above are immunoeffectors (circulatingautoantibodies or CTL clones) from patients usually having alreadyadvanced cancer. A number of data indicate that tumors can lead, forexample, to tolerization and anergization of T cells and that, duringthe course of the disease, especially those specificities which couldcause effective immune recognition are lost from the immunoeffectorrepertoire. Current patient studies have not yet produced any solidevidence of a real action of the previously found and utilizedtumor-associated antigens. Accordingly, it cannot be ruled out thatproteins evoking spontaneous immune responses are the wrong targetstructures.

SUMMARY OF THE INVENTION

It was the object of the present invention to provide target structuresfor a diagnosis and therapy of cancers.

According to the invention, this object is achieved by the subjectmatter of the claims.

According to the invention, a strategy for identifying and providingantigens expressed in association with a tumor and the nucleic acidscoding therefor was pursued. This strategy is based on the evaluation ofhuman protein and nucleic acid data bases with respect to potentialcancer-specific antigens which are accessible on the cell surface. Thedefinition of the filter criteria which are necessary for this togetherwith a high throughput methodology for analysing all proteins, ifpossible, form the central part of the invention. Data mining firstproduces a list which is as complete as possible of all known geneswhich according to the basic principle “gene to mRNA to protein” areexamined for the presence of one or more transmembrane domains. This isfollowed by a homology search, a classification of the hits in tissuespecific groups (among others tumor tissue) and an inspection of thereal existence of the mRNA. Finally, the proteins which are identifiedin this manner are evaluated for their aberrant activation in tumors,e.g. by expression analyses and protein chemical procedures.

Data mining is a known method of identifying tumor-associated genes. Inthe conventional strategies, however, transcriptoms of normal tissuelibraries are usually subtracted electronically from tumor tissuelibraries, with the assumption that the remaining genes aretumor-specific (Schmitt et al., Nucleic Acids Res. 27:4251-60, 1999;Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95:300-4, 1998; Scheurleet al., Cancer Res. 60:4037-43, 2000).

The concept of the invention, however, is based on utilizing data miningfor electronically extracting all genes coding for cancer specificantigens which are accessible on the cell surfaces and then evaluatingsaid genes for ectopic expression in tumors.

The invention thus relates in one aspect to a strategy for identifyinggenes differentially expressed in tumors. Said strategy combines datamining of public sequence libraries (“in silico”) with subsequentlaboratory-experimental (“wet bench”) studies.

According to the invention, a combined strategy based on differentbioinformatic scripts enabled new genes coding for cancer specificantigens which are accessible on the cell surfaces to be identified.According to the invention, these tumor-associated genes and the geneticproducts encoded thereby were identified and provided independently ofan immunogenic action.

The tumor-associated antigens identified according to the invention havean amino acid sequence encoded by a nucleic acid which is selected fromthe group consisting of (a) a nucleic acid which comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 69, 71, 73, 75, 79, 80,85, 87, 102, 104, 106, 108, 110, 112, a part or derivative thereof, (b)a nucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c). In a preferredembodiment, a tumor-associated antigen identified according to theinvention has an amino acid sequence encoded by a nucleic acid which isselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 69, 71, 73, 75, 79, 80, 85, 87, 102,104, 106, 108, 110, 112. In a further preferred embodiment, atumor-associated antigen identified according to the invention comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 61 to 68, 70,72, 74, 76, 81, 82, 86, 88, 96 to 101, 103, 105, 107, 109, 111, 113, apart or derivative thereof.

The present invention generally relates to the use of tumor-associatedantigens identified according to the invention or of parts thereof, ofnucleic acids coding therefor or of nucleic acids directed against saidcoding nucleic acids or of antibodies directed against thetumor-associated antigens identified according to the invention or partsthereof for therapy and diagnosis. This utilization may relate toindividual but also to combinations of two or more of these antigens,functional fragments, nucleic acids, antibodies, etc., in one embodimentalso in combination with other tumor-associated genes and antigens fordiagnosis, therapy and progress control.

The property of the tumor-associated antigens identified according tothe invention that they are localized on or at the cell surfacequalifies them as suitable targets or means for therapy and diagnosis.Especially suitable for this is a part of the tumor-associated antigensidentified according to the invention which corresponds to thenon-transmembrane portion, in particular the extracellular portion ofthe antigens, or is comprised thereof. Therefore, according to theinvention, a part of the tumor-associated antigens identified accordingto the invention which corresponds to the non-transmembrane portion ofthe antigens or is comprised thereof, or a corresponding part of thenucleic acids coding for the antigens identified according to theinvention is preferred for therapy or diagnosis. Similarly, the use ofantibodies is preferred which are directed against a part of thetumor-associated antigens identified according to the invention whichcorresponds to the non-transmembrane portion of the antigens or iscomprised thereof.

Preferred diseases for a therapy and/or diagnosis are those in which oneor more of the tumor-associated antigens identified according to theinvention are selectively expressed or abnormally expressed.

The invention also relates to nucleic acids and genetic products whichare expressed in association with a tumor cell and which are produced byaltered splicing (splice variants) of nucleic acids of thetumor-associated antigens identified according to the invention or byaltered translation with utilization of alternative open reading frames.The splice variants of the invention can be used according to theinvention as targets for diagnosis and therapy of tumor diseases.

Very different mechanisms may cause splice variants to be produced, forexample

-   -   utilization of variable transcription initiation sites    -   utilization of additional exons    -   complete or incomplete splicing out of single or two or more        exons,    -   splice regulator sequences altered via mutation (deletion or        generation of new donor/acceptor sequences),    -   incomplete elimination of intron sequences.

Altered splicing of a gene results in an altered transcript sequence(splice variant). Translation of a splice variant in the region of itsaltered sequence results in an altered protein which may be distinctlydifferent in the structure and function from the original protein.Tumor-associated splice variants may produce tumor-associatedtranscripts and tumor-associated proteins/antigens. These may beutilized as molecular markers both for detecting tumor cells and fortherapeutic targeting of tumors. Detection of tumor cells, for examplein blood, serum, bone marrow, sputum, bronchial lavage, bodilysecretions and tissue biopsies, may be carried out according to theinvention, for example, after extraction of nucleic acids by PCRamplification with splice variant-specific oligonucleotides. Accordingto the invention, all sequence-dependent detection systems are suitablefor detection. These are, apart from PCR, for example genechip/microarray systems, Northern blot, RNAse protection assays (RDA)and others. All detection systems have in common that detection is basedon a specific hybridization with at least one splice variant-specificnucleic acid sequence. However, tumor cells may also be detectedaccording to the invention by antibodies which recognize a specificepitope encoded by the splice variant. Said antibodies may be preparedby using for immunization peptides which are specific for said splicevariant. Suitable for immunization are particularly the amino acidswhose epitopes are distinctly different from the variant(s) of thegenetic product, which is (are) preferably produced in healthy cells.Detection of the tumor cells with antibodies may be carried out here ona sample isolated from the patient or as imaging with intravenouslyadministered antibodies.

In addition to diagnostic usability, splice variants having new oraltered epitopes are attractive targets for immunotherapy. The epitopesof the invention may be utilized for targeting therapeutically activemonoclonal antibodies or T lymphocytes. In passive immunotherapy,antibodies or T lymphocytes which recognize splice variant-specificepitopes are adoptively transferred here. As in the case of otherantigens, antibodies may be generated also by using standardtechnologies (immunization of animals, panning strategies for isolationof recombinant antibodies) with utilization of polypeptides whichinclude these epitopes. Alternatively, it is possible to utilize forimmunization nucleic acids coding for oligo- or polypeptides whichcontain said epitopes. Various techniques for in vitro or in vivogeneration of epitope-specific T lymphocytes are known and have beendescribed in detail (for example Kessler J H, et al. 2001, Sahin et al.,1997) and are likewise based on utilizing oligo- or polypeptides whichcontain the splice variant-specific epitopes or nucleic acids coding forsaid oligo- or polypeptides. Oligo- or polypeptides which contain thesplice variant-specific epitopes or nucleic acids coding for saidpolypeptides may also be used for utilization as pharmaceutically activesubstances in active immunotherapy (vaccination, vaccine therapy).

In a further aspect, the invention also relates to posttranslationallymodified protein domains such as glycosylations or myristoylations. Thiskind of modifications can result in a differential recognition patternof an antigen, e.g. by an antibody, and recognize different conditionspossibly associated with a disease. In particular by using antibodies,this differentiation of an antigen can be utilized diagnostically aswell as therapeutically. It has been published for tumor cells that thetumor-associated cellular degeneration can result in alteredposttranslational modifications (Durand & Seta. 2000. Clin Chem 46:795-805; Granovsky et al. 2000. Nat Med 6: 306-312). In particular,glycosylation patterns are strongly altered on tumor cells. Thesespecial epitopes according to the invention can discriminate tumor cellsfrom non-carcinogenic cells diagnostically. If an epitope which can bemodified posttranslationally is glycosylated in normal non-degeneratedcells and is deglycosylated in tumor cells, this situation makes thedevelopment of a tumor specific therapeutic antibody within the scope ofthe invention possible.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which recognizes the tumor-associated antigenidentified according to the invention and which is preferably selectivefor cells which have expression or abnormal expression of atumor-associated antigen identified according to the invention. Inparticular embodiments, said agent may cause induction of cell death,reduction in cell growth, damage to the cell membrane or secretion ofcytokines and preferably have a tumor-inhibiting activity. In oneembodiment, the agent is an antisense nucleic acid which hybridizesselectively with the nucleic acid coding for the tumor-associatedantigen. In a further embodiment, the agent is an antibody which bindsselectively to the tumor-associated antigen, in particular acomplement-activated antibody which binds selectively to thetumor-associated antigen. In a further embodiment, the agent comprisestwo or more agents which each selectively recognize differenttumor-associated antigens, at least one of which is a tumor-associatedantigen identified according to the invention. Recognition needs not beaccompanied directly with inhibition of activity or expression of theantigen. In this aspect of the invention, the antigen selectivelylimited to tumors preferably serves as a label for recruiting effectormechanisms to this specific location. In a preferred embodiment, theagent is a cytotoxic T lymphocyte which recognizes the antigen on an HLAmolecule and lyses the cell labeled in this way. In a furtherembodiment, the agent is an antibody which binds selectively to thetumor-associated antigen and thus recruits natural or artificialeffector mechanisms to said cell. In a further embodiment, the agent isa T helper lymphocyte which enhances effector functions of other cellsspecifically recognizing said antigen.

In one aspect, the invention relates to a pharmaceutical compositioncomprising an agent which inhibits expression or activity of atumor-associated antigen identified according to the invention. In apreferred embodiment, the agent is an antisense nucleic acid whichhybridizes selectively with the nucleic acid coding for thetumor-associated antigen. In a further embodiment, the agent is anantibody which binds selectively to the tumor-associated antigen. In afurther embodiment, the agent comprises two or more agents which eachselectively inhibit expression or activity of different tumor-associatedantigens, at least one of which is a tumor-associated antigen identifiedaccording to the invention.

The activity of a tumor-associated antigen identified according to theinvention can be any activity of a protein or a peptide. Thus, thetherapeutic and diagnostic methods according to the invention can alsoaim at inhibiting or reducing this activity or testing this activity.

The invention furthermore relates to a pharmaceutical composition whichcomprises an agent which, when administered, selectively increases theamount of complexes between an HLA molecule and a peptide epitope fromthe tumor-associated antigen identified according to the invention. Inone embodiment, the agent comprises one or more components selected fromthe group consisting of (i) the tumor-associated antigen or a partthereof, (ii) a nucleic acid which codes for said tumor-associatedantigen or a part thereof, (iii) a host cell which expresses saidtumor-associated antigen or a part thereof, and (iv) isolated complexesbetween peptide epitopes from said tumor-associated antigen and an MHCmolecule. In one embodiment, the agent comprises two or more agentswhich each selectively increase the amount of complexes between MHCmolecules and peptide epitopes of different tumor-associated antigens,at least one of which is a tumor-associated antigen identified accordingto the invention.

The invention furthermore relates to a pharmaceutical composition whichcomprises one or more components selected from the group consisting of(i) a tumor-associated antigen identified according to the invention ora part thereof, (ii) a nucleic acid which codes for a tumor-associatedantigen identified according to the invention or for a part thereof,(iii) an antibody which binds to a tumor-associated antigen identifiedaccording to the invention or to a part thereof, (iv) an antisensenucleic acid which hybridizes specifically with a nucleic acid codingfor a tumor-associated antigen identified according to the invention,(v) a host cell which expresses a tumor-associated antigen identifiedaccording to the invention or a part thereof, and (vi) isolatedcomplexes between a tumor-associated antigen identified according to theinvention or a part thereof and an HLA molecule.

A nucleic acid coding for a tumor-associated antigen identifiedaccording to the invention or for a part thereof may be present in thepharmaceutical composition in an expression vector and functionallylinked to a promoter.

A host cell present in a pharmaceutical composition of the invention maysecrete the tumor-associated antigen or the part thereof, express it onthe surface or may additionally express an HLA molecule which binds tosaid tumor-associated antigen or said part thereof. In one embodiment,the host cell expresses the HLA molecule endogenously. In a furtherembodiment, the host cell expresses the HLA molecule and/or thetumor-associated antigen or the part thereof in a recombinant manner.The host cell is preferably nonproliferative. In a preferred embodiment,the host cell is an antigen-presenting cell, in particular a dendriticcell, a monocyte or a macrophage.

An antibody present in a pharmaceutical composition of the invention maybe a monoclonal antibody. In further embodiments, the antibody is achimeric or humanized antibody, a fragment of a natural antibody or asynthetic antibody, all of which may be produced by combinatorytechniques. The antibody may be coupled to a therapeutically ordiagnostically useful agent.

An antisense nucleic acid present in a pharmaceutical composition of theinvention may comprise a sequence of 6-50, in particular 10-30, 15-30and 20-30, contiguous nucleotides of the nucleic acid coding for thetumor-associated antigen identified according to the invention.

In further embodiments, a tumor-associated antigen, provided by apharmaceutical composition of the invention either directly or viaexpression of a nucleic acid, or a part thereof binds to MHC moleculeson the surface of cells, said binding preferably causing a cytolyticresponse and/or inducing cytokine release.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier and/or an adjuvant. The adjuvant maybe selected from saponin, GM-CSF, CpG oligonucleotides, RNA, a cytokineor a chemokine. A pharmaceutical composition of the invention ispreferably used for the treatment of a disease characterized byselective expression or abnormal expression of a tumor-associatedantigen. In a preferred embodiment, the disease is cancer.

The invention furthermore relates to methods of treating or diagnosing adisease characterized by expression or abnormal expression of one ofmore tumor-associated antigens. In one embodiment, the treatmentcomprises administering a pharmaceutical composition of the invention.

In one aspect, the invention relates to a method of diagnosing a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention. The method comprises (i)detection of a nucleic acid which codes for the tumor-associated antigenor of a part thereof and/or (ii) detection of the tumor-associatedantigen or of a part thereof, and/or (iii) detection of an antibody tothe tumor-associated antigen or to a part thereof and/or (iv) detectionof cytotoxic or T helper lymphocytes which are specific for thetumor-associated antigen or for a part thereof in a biological sampleisolated from a patient. In particular embodiments, detection comprises(i) contacting the biological sample with an agent which bindsspecifically to the nucleic acid coding for the tumor-associated antigenor to the part thereof, to said tumor-associated antigen or said partthereof, to the antibody or to cytotoxic or T helper lymphocytesspecific for the tumor-associated antigen or parts thereof, and (ii)detecting the formation of a complex between the agent and the nucleicacid or the part thereof, the tumor-associated antigen or the partthereof, the antibody or the cytotoxic or T helper lymphocytes. In oneembodiment, the disease is characterized by expression or abnormalexpression of two or more different tumor-associated antigens anddetection comprises detection of two or more nucleic acids coding forsaid two or more different tumor-associated antigens or of partsthereof, detection of two or more different tumor-associated antigens orof parts thereof, detection of two or more antibodies binding to saidtwo or more different tumor-associated antigens or to parts thereof ordetection of two or more cytotoxic or T helper lymphocytes specific forsaid two or more different tumor-associated antigens. In a furtherembodiment, the biological sample isolated from the patient is comparedto a comparable normal biological sample.

The methods of diagnosing according to the invention can concern alsothe use of the tumor-associated antigens identified according to theinvention as prognostic markers, in order to predict metastasis, e.g.through testing the migration behavior of cells, and therefore aworsened course of the disease, whereby among other things planning of amore aggressive therapy is made possible.

In a further aspect, the invention relates to a method for determiningregression, course or onset of a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises monitoring a sample from apatient who has said disease or is suspected of falling ill with saiddisease, with respect to one or more parameters selected from the groupconsisting of (i) the amount of nucleic acid which codes for thetumor-associated antigen or of a part thereof, (ii) the amount of thetumor-associated antigen or a part thereof, (iii) the amount ofantibodies which bind to the tumor-associated antigen or to a partthereof, and (iv) the amount of cytolytic T cells or T helper cellswhich are specific for a complex between the tumor-associated antigen ora part thereof and an MHC molecule. The method preferably comprisesdetermining the parameter(s) in a first sample at a first point in timeand in a further sample at a second point in time and in which thecourse of the disease is determined by comparing the two samples. Inparticular embodiments, the disease is characterized by expression orabnormal expression of two or more different tumor-associated antigensand monitoring comprises monitoring (i) the amount of two or morenucleic acids which code for said two or more different tumor-associatedantigens or of parts thereof, and/or (ii) the amount of said two or moredifferent tumor-associated antigens or of parts thereof, and/or (iii)the amount of two or more antibodies which bind to said two or moredifferent tumor-associated antigens or to parts thereof, and/or (iv) theamount of two or more cytolytic T cells or of T helper cells which arespecific for complexes between said two or more differenttumor-associated antigens or of parts thereof and MHC molecules.

According to the invention, detection of a nucleic acid or of a partthereof or monitoring the amount of a nucleic acid or of a part thereofmay be carried out using a polynucleotide probe which hybridizesspecifically to said nucleic acid or said part thereof or may be carriedout by selective amplification of said nucleic acid or said partthereof. In one embodiment, the polynucleotide probe comprises asequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguousnucleotides of said nucleic acid.

According to the invention, detection of a tumor-associated antigen orof a part thereof or monitoring the amount of a tumor-associated antigenor of a part thereof may be carried out using an antibody bindingspecifically to said tumor-associated antigen or said part thereof.

In certain embodiments, the tumor-associated antigen to be detected orthe part thereof is present in a complex with an MHC molecule, inparticular an HLA molecule.

According to the invention, detection of an antibody or monitoring theamount of antibodies may be carried out using a protein or peptidebinding specifically to said antibody.

According to the invention, detection of cytolytic T cells or of Thelper cells or monitoring the amount of cytolytic T cells or of Thelper cells which are specific for complexes between an antigen or apart thereof and MHC molecules may be carried out using a cellpresenting the complex between said antigen or said part thereof and anMHC molecule.

The polynucleotide probe, the antibody, the protein or peptide or thecell, which is used for detection or monitoring, is preferably labeledin a detectable manner. In particular embodiments, the detectable markeris a radioactive marker or an enzymic marker. T lymphocytes mayadditionally be detected by detecting their proliferation, theircytokine production, and their cytotoxic activity triggered by specificstimulation with the complex of MHC and tumor-associated antigen orparts thereof. T lymphocytes may also be detected via a recombinant MHCmolecule or else a complex of two or more MHC molecules which are loadedwith the particular immunogenic fragment of one or more of thetumor-associated antigens and by contacting the specific T cell receptorwhich can identify the specific T lymphocytes.

In a further aspect, the invention relates to a method of treating,diagnosing or monitoring a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the invention, which method comprises administering an antibody whichbinds to said tumor-associated antigen or to a part thereof and which iscoupled to a therapeutic or diagnostic agent. The antibody may be amonoclonal antibody. In further embodiments, the antibody is a chimericor humanized antibody or a fragment of a natural antibody.

The invention also relates to a method of treating a patient having adisease characterized by expression or abnormal expression of atumor-associated antigen identified according to the invention, whichmethod comprises (i) removing a sample containing immunoreactive cellsfrom said patient, (ii) contacting said sample with a host cellexpressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic T cells against saidtumor-associated antigen or a part thereof, and (iii) introducing thecytolytic T cells into the patient in an amount suitable for lysingcells expressing the tumor-associated antigen or a part thereof. Theinvention likewise relates to cloning the T cell receptor of cytolytic Tcells against the tumor-associated antigen. Said receptor may betransferred to other T cells which thus receive the desired specificityand, as under (iii), may be introduced into the patient.

In one embodiment, the host cell endogenously expresses an HLA molecule.In a further embodiment, the host cell recombinantly expresses an HLAmolecule and/or the tumor-associated antigen or the part thereof. Thehost cell is preferably nonproliferative. In a preferred embodiment, thehost cell is an antigen-presenting cell, in particular a dendritic cell,a monocyte or a macrophage.

In a further aspect, the invention relates to a method of treating apatient having a disease characterized by expression or abnormalexpression of a tumor-associated antigen, which method comprises (i)identifying a nucleic acid which codes for a tumor-associated antigenidentified according to the invention and which is expressed by cellsassociated with said disease, (ii) transfecting a host cell with saidnucleic acid or a part thereof, (iii) culturing the transfected hostcell for expression of said nucleic acid (this is not obligatory when ahigh rate of transfection is obtained), and (iv) introducing the hostcells or an extract thereof into the patient in an amount suitable forincreasing the immune response to the patient's cells associated withthe disease. The method may further comprise identifying an MHC moleculepresenting the tumor-associated antigen or a part thereof, with the hostcell expressing the identified MHC molecule and presenting saidtumor-associated antigen or a part thereof. The immune response maycomprise a B cell response or a T cell response. Furthermore, a T cellresponse may comprise production of cytolytic T cells and/or T helpercells which are specific for the host cells presenting thetumor-associated antigen or a part thereof or specific for cells of thepatient which express said tumor-associated antigen or a part thereof.

The invention also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the invention, which method comprises(i) identifying cells from the patient which express abnormal amounts ofthe tumor-associated antigen, (ii) isolating a sample of said cells,(iii) culturing said cells, and (iv) introducing said cells into thepatient in an amount suitable for triggering an immune response to thecells.

Preferably, the host cells used according to the invention arenonproliferative or are rendered nonproliferative. A diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen is in particular cancer.

The present invention furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:69, 71, 73, 79, 80, 85, 87, 102, 104, 106, 108, 110, 112, a part orderivative thereof, (b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, (c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c). The invention furthermore relates to a nucleic acid, which codesfor a protein or polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 61-68, 70, 72, 74, 81, 82, 86,88, 96-101, 103, 105, 107, 109, 111, 113, a part or derivative thereof.

In a further aspect, the invention relates to a recombinant nucleic acidmolecule, in particular DNA or RNA molecule, which comprises a nucleicacid of the invention.

The invention also relates to host cells which contain a nucleic acid ofthe invention or a recombinant nucleic acid molecule comprising anucleic acid of the invention.

The host cell may also comprise a nucleic acid coding for a HLAmolecule. In one embodiment, the host cell endogenously expresses theHLA molecule. In a further embodiment, the host cell recombinantlyexpresses the HLA molecule and/or the nucleic acid of the invention or apart thereof. Preferably, the host cell is nonproliferative. In apreferred embodiment, the host cell is an antigen-presenting cell, inparticular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, the invention relates to oligonucleotides whichhybridize with a nucleic acid identified according to the invention andwhich may be used as genetic probes or as “antisense” molecules. Nucleicacid molecules in the form of oligonucleotide primers or competentsamples, which hybridize with a nucleic acid identified according to theinvention or parts thereof, may be used for finding nucleic acids whichare homologous to said nucleic acid identified according to theinvention. PCR amplification, Southern and Northern hybridization may beemployed for finding homologous nucleic acids. Hybridization may becarried out under low stringency, more preferably under mediumstringency and most preferably under high stringency conditions. Theterm “stringent conditions” according to the invention refers toconditions which allow specific hybridization between polynucleotides.

In a further aspect, the invention relates to a protein or polypeptidewhich is encoded by a nucleic acid selected from the group consisting of(a) a nucleic acid which comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 69, 71, 73, 79, 80, 85, 87, 102,104, 106, 108, 110, 112, a part or derivative thereof, (b) a nucleicacid which hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c). In a preferredembodiment, the invention relates to a protein or polypeptide whichcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 61-68, 70, 72, 74, 81, 82, 86, 88, 96-101, 103, 105, 107,109, 111, 113, a part or derivative thereof.

In a further aspect, the invention relates to an immunogenic fragment ofa tumor-associated antigen identified according to the invention. Saidfragment preferably binds to a human HLA receptor or to a humanantibody. A fragment of the invention preferably comprises a sequence ofat least 6, in particular at least 8, at least 10, at least 12, at least15, at least 20, at least 30 or at least 50, amino acids. In particularan immunogenic fragment according to the invention comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 61-68,81, 82, and 96-101, a part or derivative thereof.

In a further aspect, the invention relates to an agent which binds to atumor-associated antigen identified according to the invention or to apart thereof. In a preferred embodiment, the agent is an antibody. Infurther embodiments, the antibody is a chimeric, a humanized antibody oran antibody produced by combinatory techniques or is a fragment of anantibody. Furthermore, the invention relates to an antibody which bindsselectively to a complex of (i) a tumor-associated antigen identifiedaccording to the invention or a part thereof and (ii) an MHC molecule towhich said tumor-associated antigen identified according to theinvention or said part thereof binds, with said antibody not binding to(i) or (ii) alone. An antibody of the invention may be a monoclonalantibody. In further embodiments, the antibody is a chimeric orhumanized antibody or a fragment of a natural antibody.

The invention furthermore relates to a conjugate between an agent of theinvention which binds to a tumor-associated antigen identified accordingto the invention or to a part thereof or an antibody of the inventionand a therapeutic or diagnostic agent. In one embodiment, thetherapeutic or diagnostic agent is a toxin.

In a further aspect, the invention relates to a kit for detectingexpression or abnormal expression of a tumor-associated antigenidentified according to the invention, which kit comprises agents fordetection (i) of the nucleic acid which codes for the tumor-associatedantigen or of a part thereof, (ii) of the tumor-associated antigen or ofa part thereof, (iii) of antibodies which bind to the tumor-associatedantigen or to a part thereof, and/or (iv) of T cells which are specificfor a complex between the tumor-associated antigen or a part thereof andan MHC molecule. In one embodiment, the agents for detection of thenucleic acid or the part thereof are nucleic acid molecules forselective amplification of said nucleic acid, which comprise, inparticular a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of said nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Quantitative expression analysis of FLJ31461 in normal tissues(left) and in various tumors (pools consisting of 3-4 individual sampleseach, right) in a logarithmic representation of the relative expression(x-fold activation). In most tumors and at least 100-fold overexpressionof FLJ31461 is observed in comparison to the level of expression inhealthy tissues.

FIG. 1B: Gel image of a conventional RT-PCR-analysis of FLJ31461 intumors of the breast, lungs and ear, nose and throat with theappropriate normal tissues N_(x); M: DNA-length marker.

FIG. 1C: Quantitative expression analysis in various normal tissues(left) and in breast tumors in a logarithmic representation of therelative expression (x-fold activation). In almost all breast tumors andat least 100-fold overexpression of FLJ31461 is observed in comparisonto the level of expression in healthy tissues.

FIG. 1D: Summary of the FLJ31461-specific expression in various analysedtumors. Shown is the number of positively tested tumor samples relativeto the total number of analysed tumor samples. While all investigatednormal somatic tissues (3-10 tissues each, depending on tissue type)exhibit no expression of FLJ31461, the gene is expressed in many tumorswith variable frequency.

FIG. 2: Representation of the cellular localisation of theFLJ31461-protein. FIG. 2 shows the endogenous protein expression of thebreast tumor cell-line MCF7.

FIG. 3A: Normal tissue of testis (positive membrane localisation), colonand kidney (negative membrane localisation).

FIG. 3B: Detection of the FLJ31461-protein in a bronchial carcinoma, acervical carcinoma as well as a lymphatic node metastasis of a breasttumor in an overview (left column) and in detail (right column).

FIG. 3C: Summary of the immunohistochemical analyses of theFLJ31461-protein. Shown is the number of positively tested tumor samplesin relation to the total number of analysed tumor samples. While allinvestigated normal somatic tissues did not exhibit any expression ofFLJ31461, the protein is detected in many of the tumors with varyingfrequency at the cell surface.

FIG. 4A: The PCR on normal tissues and various tumors was carried outusing DSG4-specific oligonucleotides in exons 8-12 and exons 10-12. Thedominant expression of the transcript of exons 10-12 is recognisable incolon tumors, while the transcript of exons 8-12 is also clearlyexpressed in normal tissues. Ge: brain, Dd: duodenum, Pa: pancreas, Mi:spleen, Te: testis, He: heart, Ko: colon, LN: lymphatic node, TM:thymus, Pr: prostate, Ös: esophagus, Le: liver, PB: active PBMC, Lu:lung, Bl: bladder, Ma: stomach, Br: breast, Ut: uterus, Ov: ovary, Ni:kidney, Ha: skin, Mu: muscle.

FIG. 4B: Summary of the specific expression of the DSG4-exons 10-12 invarious analysed tumors. Shown is the number of positively tested tumorsamples relative to the total number. While almost all investigatednormal somatic tissues did not exhibit any expression of DSG4, thisgene-section is detectable in many of the tumors with varying frequency.

FIG. 4C: Quantitative expression analysis of the transcript section ofthe DSG4-exons 10-12 in normal tissues (left) and in tumors of thecolon, stomach and the ear-nose-throat area in logarithmicrepresentation of relative expression (x-fold activation). Most tumorsexhibited an at least 50-fold over-expression of the DSG4-exons 10-12 incomparison to the expression levels in healthy tissues.

FIG. 5: Overview of the putative transcript variants of the DSG4-gene.

FIG. 6A: Representation of the cellular localisation of the DSG4-proteinusing immunofluorescence on a DSG4-transfected cell.

FIG. 6B: FACS-analysis of DSG4-transfected cells with DSG4-specificantibodies (left figure) and of Mock-transfected cells withDSG4-specific antibodies (negative control, right figure). The specific,surface-specific staining is clearly visible.

FIG. 7A: Quantitative expression analysis of DSG3 in normal tissues(left side) and in various tumors (pools consisting of 3-4 individualsamples each, right side) in logarithmic representation of the relativeexpression (x-fold activation). The distinct overexpression inesophageal tumors in comparison to most normal tissues is recognisable.

FIG. 7B: Quantitative expression analysis of DSG3 in various tumors ofthe cervix and lungs as well as in ear, nose, throat tumors incomparison to the expression in the respective normal tissues (n=3(cervix); n=9 (lung)). Logarithmic representation.

FIG. 7C: Summary of the DSG3-specific expression in various analysedtumors. Shown is the number of positively tested tumor samples relativeto the total number of analysed tumor samples. While all investigatednormal somatic tissues (3-10 tissues each, depending on tissue type) donot show any expression of DSG3, the gene is expressed in many tumorswith varying frequency.

FIG. 8: Shows in an overview (left) and in detail (right) the homogenousDSG3-localisation in an ear, nose, throat tumor.

FIG. 9A: Quantitative expression analysis of SLC6A3 in normal tissues(left) and in tumor samples (pools consisting of 3-4 individual sampleseach, right) in logarithmic representation of the relative expression(x-fold activation).

FIG. 9B: Quantitative expression analysis of SLC6A3 in various kidneytumors in comparison to the expression in normal kidney (n=5).Logarithmic representation of the relative expression.

FIG. 9C: Conventional endpoint-RT-PCR-analysis of SLC6A3-specifictranscripts (double determination) in kidney tumors and various normalkidney tissues. Image after gel-electrophoretic resolution of theSLC6A3-specific fragments.

FIG. 9D: Quantitative expression analysis of SLC6A3 in carcinomas of thebreast, ovary, lung and prostate; Logarithmic representation of therelative expression (x-fold activation). “Tissue” N: normal tissue;“Tissue”: tumor tissue.

FIG. 9E: Conventional RT-PCR-analysis of SLC6A3 in tumors of the breast,ovary, lung and prostate after gel-electrophoretic separation in adouble determination. M: DNA-length marker.

FIG. 10A: Quantitative expression analysis of GRM8 in normal tissues(left) and tumor tissues (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 10B: Quantitative expression analysis of GRM8 in various tumors ofthe kidney and uterus in comparison to the expression in the normalkidney and uterus, as well as relative expression in ear, nose, throattumors, cervical tumors and melanomas. Logarithmic representation of therelative expression.

FIG. 11A: Quantitative expression analysis of CDH17 in normal tissues(left) and in tumor tissues (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 11B: Quantitative expression analysis of CDH17 in various tumors ofthe colon and stomach in comparison to the expression in the respectivenormal tissues. Logarithmic representation.

FIG. 11C: Quantitative expression analysis of CDH17 in various tumors ofthe esophagus and pancreas in comparison to the expression in therespective normal tissues. Logarithmic representation.

FIG. 12: Shows quantitative expression analysis of ABCC4 in normaltissues (left) and tumors (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 13A: Quantitative expression analysis of VIL1 in normal tissues(left) and tumor tissues (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 13B: Quantitative expression analysis of VIL1 in various tumors ofthe colon and stomach in comparison to the expression in the respectivenormal tissues. Logarithmic representation.

FIG. 14A: Quantitative expression analysis of MGC34032 in normal tissues(left) and various tumors (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 14B: Quantitative expression analysis of MGC34032 in various tumorsof the esophagus, pancreas and colon in comparison to the expression inthe respective normal tissues. Logarithmic representation.

FIG. 14C: Quantitative expression analysis of MGC34032 in various tumorsof the lung, ovary and kidney in comparison to the expression in therespective normal tissues. Logarithmic representation.

FIG. 14D: Summary of the MGC34032-specific expression in variousanalysed tumors. Shown is the number of positively tested tumor samplesrelative to the total number of the analysed tumor samples. While allinvestigated somatic normal tissues (3-10 tissues each, depending ontissue type) exhibit a significantly lower expression of MGC34032, thegene is overexpressed in many tumors with varying frequency.

FIG. 15: Shows 2 detailed views of the cellular localisation of theMGC34032-protein in human testis tissue.

FIG. 16A: Quantitative expression analysis of PRSS7 in normal tissues(left) and various tumor tissues (pools consisting of 3-4 individualsamples each, right) in linear representation of the relative expression(x-fold activation).

FIG. 16B: Quantitative expression analysis of PRSS7 in various tumors ofthe stomach and esophagus in comparison to the expression in therespective normal tissues (stomach: n=7; esophagus: n=3). For comparisonthe expression was measured in a normal duodenum (n=2). Logarithmicrepresentation.

FIG. 16C: Quantitative expression analysis of PRSS7 in various pancreasand liver tumors in comparison to the expression in the respectivenormal tissues (n=4 for each). For comparison the expression in normalduodenum was measured (n=2). Logarithmic representation.

FIG. 17A: Representation of the cellular localisation of thePRSS7-protein on PRSS7-transfected cells.

FIG. 17B: Detection of the PRSS7-protein in overview (left) and indetail (right).

FIG. 18A: Quantitative expression analysis of CLCA2 in normal tissues(left) and various tumors (pools consisting of 3-4 individual sampleseach, right) in logarithmic representation of the relative expression(x-fold activation).

FIG. 18B: Quantitative expression analysis of CLCA2 in various tumors ofthe lung, breast, cervix and uterus and in ear, nose and throat tumorsin comparison to the expression in the respective normal tissues.Logarithmic representation.

FIG. 18C: Summary of the CLCA2-specific expression in various analysedtumors. Shown is the number of positively tested tumor samples relativeto the number of total samples of analysed tumors. While allinvestigated normal somatic tissues exhibit a significantly lowerexpression of CLCA2, the gene is overexpressed in many tumors withvarying frequency.

FIG. 19A: Representation of the localisation of the CLCA2-protein at themembrane of CLCA2-transfected cells.

FIG. 19B: The figure shows the immunohistochemical analysis at theCLCA2-protein.

FIG. 20A: Quantitative expression analysis of TM4SF4 in normal tissues(left) and in various tumors (pools consisting of 3-4 individual sampleseach, right) in linear representation of the relative expression (x-foldactivation).

FIG. 20B: Quantitative expression analysis of TM4SF4 in various livertumors in comparison to 4 different normal tissues of the liver (N0 toN3); linear representation.

FIG. 20C: Logarithmic representation of the relative expression ofTM4SF4 in 12 different colon tumors in comparison to normal colonsamples (NG: normal tissue; 6 different normal tissues wereinvestigated).

FIG. 21A: The image shows an immunoblot with TM4SF4-specific antibodiesin normal liver tissue and liver tumor tissue. Two putativeglycosylation parameters are recognisable.

FIG. 21B: The figure shows the localisation of the TM4SF4-protein at themembrane of TM4SF4-transfected cells.

FIG. 21C: The immunohistochemical analysis was able to confirm theexpression selectivity observed by PCR.

FIG. 22A: Quantitative expression analysis of claudin19 in normaltissues (left) and in various tumors (pools consisting of 3-4 individualsamples each, right) in logarithmic representation of the relativeexpression (x-fold activation).

FIG. 22B: Quantitative expression analysis of claudin19 in variousbreast tumors and the respective normal breast tissues.

FIG. 22C: Conventional RT-PCR with analysis of claudin19 in variousbreast tumor samples as well as in a normal tissue; M: DNA-lengthmarker.

FIG. 22D: Conventional RT-PCR-analysis of claudin19 in various normaltissues of the stomach and stomach tumors.

FIG. 22E: Conventional RT-PCR-analysis of claudin19 in various normaltissues of the liver and liver tumors; M: DNA-length marker.

FIG. 23A: Quantitative expression analysis of ALPPL2 in normal tissues(left) and in tumors (pools consisting of 3-4 individual samples each,right) in linear determination of the relative expression (x-foldactivation).

FIG. 23B: Gel image of a conventional RT-PCR-analysis of ALPPL2 invarious tumors of the colon and stomach as well as in the respectivenormal tissues after gel-electrophoretic separation; M: DNA-lengthmarker.

FIG. 24A: Quantitative expression analysis of GPR64 in normal tissues(left) and in tumors (pools consisting of 3-4 individual samples each,right) in linear representation of the relative expression (x-foldactivation).

FIG. 24B: Quantitative expression analysis of GPR64 in various tumors ofthe ovary and the respective normal ovary tissues.

FIG. 24C: Gel-image of a RT-PCR-analysis of GPR64 in various tumors ofthe ovary and in normal tissues; M: DNA-length marker.

FIG. 25A: Quantitative expression analysis of SLC12A1 in normal tissues(left) and in tumors (pools consisting of 3-4 individual samples, right)in linear representation of the relative expression (x-fold activation).

FIG. 25B: Quantitative expression analysis of SLC12A1 in 12 differentkidney tumors in comparison to the expression in the normal kidney(n=3).

FIG. 25C: Quantitative expression analysis of SLC12A1 in tumors of thebreast, ovary and prostate in comparison to the expression in therespective normal tissues (breast: n=9, ovary: n=8, prostate: n=3).Logarithmic representation.

FIG. 25D: Conventional RT-PCR-analysis of SLC12A1 in kidney tumors,various normal kidneys and various tumor types (breast, prostate, ovary)with the respective normal tissues.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, genes are described which are expressed intumor cells selectively or aberrantly and which are tumor-associatedantigens.

According to the invention, these genes or their derivatives arepreferred target structures for therapeutic approaches. Conceptionally,said therapeutic approaches may aim at inhibiting the activity of theselectively expressed tumor-associated genetic product. This is useful,if said aberrant respective selective expression is functionallyimportant in tumor pathogenecity and if its ligation is accompanied byselective damage of the corresponding cells. Other therapeutic conceptscontemplate tumor-associated antigens as labels which recruit effectormechanisms having cell-damaging potential selectively to tumor cells.Here, the function of the target molecule itself and its role in tumordevelopment are totally irrelevant.

“Derivative” of a nucleic acid means according to the invention thatsingle or multiple nucleotide substitutions, deletions and/or additionsare present in said nucleic acid. Furthermore, the term “derivative”also comprises chemical derivatization of a nucleic acid on a base, on asugar or on a phosphate of a nucleotide. The term “derivative” alsocomprises nucleic acids which contain nucleotides and nucleotide analogsnot occurring naturally.

According to the invention, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the invention genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the invention, a nucleic acid may be present as a single-stranded ordouble-stranded and linear or covalently circularly closed molecule.

The nucleic acids described according to the invention have preferablybeen isolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid was (i) amplified in vitro, for exampleby polymerase chain reaction (PCR), (ii) recombinantly produced bycloning, (iii) purified, for example by cleavage and gel-electrophoreticfractionation, or (iv) synthesized, for example by chemical synthesis.An isolated nucleic acid is a nucleic acid which is available formanipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

According to the invention, complementary nucleic acids have at least40%, in particular at least 50%, at least 60%, at least 70%, at least80%, at least 90% and preferably at least 95%, at least 98% or at least99%, identical nucleotides.

Nucleic acids coding for tumor-associated antigens may, according to theinvention, be present alone or in combination with other nucleic acids,in particular heterologous nucleic acids. In preferred embodiments, anucleic acid is functionally linked to expression control sequences orregulatory sequences which may be homologous or heterologous withrespect to said nucleic acid. A coding sequence and a regulatorysequence are “functionally” linked to one another, if they arecovalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the invention promoters, enhancers and othercontrol elements which regulate expression of a gene. In particularembodiments of the invention, the expression control sequences can beregulated. The exact structure of regulatory sequences may vary as afunction of the species or cell type, but generally comprises5′untranscribed and 5′untranslated sequences which are involved ininitiation of transcription and translation, respectively, such as TATAbox, capping sequence, CAAT sequence, and the like. More specifically,5′untranscribed regulatory sequences comprise a promoter region whichincludes a promoter sequence for transcriptional control of thefunctionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

Thus, on the one hand, the tumor-associated antigens illustrated hereinmay be combined with any expression control sequences and promoters. Onthe other hand, however, the promoters of the tumor-associated geneticproducts illustrated herein may, according to the invention, be combinedwith any other genes. This allows the selective activity of thesepromoters to be utilized.

According to the invention, a nucleic acid may furthermore be present incombination with another nucleic acid which codes for a polypeptidecontrolling secretion of the protein or polypeptide encoded by saidnucleic acid from a host cell. According to the invention, a nucleicacid may also be present in combination with another nucleic acid whichcodes for a polypeptide causing the encoded protein or polypeptide to beanchored on the cell membrane of the host cell or compartmentalized intoparticular organelles of said cell.

In a preferred embodiment, a recombinant DNA molecule is according tothe invention a vector, where appropriate with a promoter, whichcontrols expression of a nucleic acid, for example a nucleic acid codingfor a tumor-associated antigen of the invention. The term “vector” isused here in its most general meaning and comprises any intermediaryvehicle for a nucleic acid which enables said nucleic acid, for example,to be introduced into prokaryotic and/or eukaryotic cells and, whereappropriate, to be integrated into a genome. Vectors of this kind arepreferably replicated and/or expressed in the cells. An intermediaryvehicle may be adapted, for example, to the use in electroporation, inbombardment with microprojectiles, in liposomal administration, in thetransfer with the aid of agrobacteria or in insertion via DNA or RNAviruses. Vectors comprise plasmids, phagemids, bacteriophages or viralgenomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the invention may be used for transfection of host cells.Nucleic acids here mean both recombinant DNA and RNA. Recombinant RNAmay be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application. According to the invention, theterm “host cell” relates to any cell which can be transformed ortransfected with an exogenous nucleic acid. The term “host cells”comprises according to the invention prokaryotic (e.g. E. coli) oreukaryotic cells (e.g. dendritic cells, B cells, CHO cells, COS cells,K562 cells, yeast cells and insect cells). Particular preference isgiven to mammalian cells such as cells from humans, mice, hamsters,pigs, goats, primates. The cells may be derived from a multiplicity oftissue types and comprise primary cells and cell lines. Specificexamples comprise keratinocytes, peripheral blood leukocytes, stem cellsof the bone marrow and embryonic stem cells. In further embodiments, thehost cell is an antigen-presenting cell, in particular a dendritic cell,monocyte or a macrophage. A nucleic acid may be present in the host cellin the form of a single copy or of two or more copies and, in oneembodiment, is expressed in the host cell.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selective markersuch as a gene imparting resistance to G418 (and thus enabling stablytransfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the invention in which an HLA molecule presents atumor-associated antigen or a part thereof, an expression vector mayalso comprise a nucleic acid sequence coding for said HLA molecule. Thenucleic acid sequence coding for the HLA molecule may be present on thesame expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe HLA molecule, both nucleic acids coding therefor are transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the HLA molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The invention also comprises kits for amplification of a nucleic acidcoding for a tumor-associated antigen. Such kits comprise, for example,a pair of amplification primers which hybridize to the nucleic acidcoding for the tumor-associated antigen. The primers preferably comprisea sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguousnucleotides of the nucleic acid and are nonoverlapping, in order toavoid the formation of primer dimers. One of the primers will hybridizeto one strand of the nucleic acid coding for the tumor-associatedantigen, and the other primer will hybridize to the complementary strandin an arrangement which allows amplification of the nucleic acid codingfor the tumor-associated antigen.

“Antisense” molecules or “antisense” nucleic acids may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the invention to an oligonucleotide which is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide or modifiedoligodeoxyribonucleotide and which hybridizes under physiologicalconditions to DNA comprising a particular gene or to mRNA of said gene,thereby inhibiting transcription of said gene and/or translation of saidmRNA. According to the invention, the “antisense molecule” alsocomprises a construct which contains a nucleic acid or a part thereof inreverse orientation with respect to its natural promoter. An antisensetranscript of a nucleic acid or of a part thereof may form a duplex withthe naturally occurring mRNA specifying the enzyme and thus preventaccumulation of or translation of the mRNA into the active enzyme.Another possibility is the use of ribozymes for inactivating a nucleicacid. Antisense oligonucleotides preferred according to the inventionhave a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of the target nucleic acid and preferably arefully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′untranslated region ormRNA splicing site.

In one embodiment, an oligonucleotide of the invention consists ofribonucleotides, deoxyribonucleotides or a combination thereof, with the5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the invention is a“modified” oligonucleotide. Here, the oligonucleotide may be modified invery different ways, without impairing its ability to bind its target,in order to increase, for example, its stability or therapeuticefficacy. According to the invention, the term “modifiedoligonucleotide” means an oligonucleotide in which (i) at least two ofits nucleotides are linked to one another by a synthetic intemucleosidebond (i.e. an intemucleoside bond which is not a phosphodiester bond)and/or (ii) a chemical group which is usually not found in nucleic acidsis covalently linked to the oligonucleotide. Preferred syntheticintemucleoside bonds are phosphorothioates, alkyl phosphonates,phosphorodithioates, phosphate esters, alkyl phosphonothioates,phosphoramidates, carbamates, carbonates, phosphate triesters,acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

Preferably, the proteins and polypeptides described according to theinvention have been isolated. The terms “isolated protein” or “isolatedpolypeptide” mean that the protein or polypeptide has been separatedfrom its natural environment. An isolated protein or polypeptide may bein an essentially purified state. The term “essentially purified” meansthat the protein or polypeptide is essentially free of other substanceswith which it is associated in nature or in vivo.

Such proteins and polypeptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and polypeptides described according to theinvention may be isolated from biological samples such as tissue or cellhomogenates and may also be expressed recombinantly in a multiplicity ofpro- or eukaryotic expression systems.

For the purposes of the present invention, “derivatives” of a protein orpolypeptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible. Amino acid deletion variants are characterized by theremoval of one or more amino acids from the sequence. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpolypeptides. Preference is given to replacing amino acids with otherones having similar properties such as hydrophobicity, hydrophilicity,electronegativity, volume of the side chain and the like (conservativesubstitution). Conservative substitutions, for example, relate to theexchange of one amino acid with another amino acid listed below in thesame group as the amino acid to be substituted:

-   -   1. small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly)    -   2. negatively charged residues and their amides: Asn, Asp, Glu,        Gln    -   3. positively charged residues: His, Arg, Lys    -   4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)    -   5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residuesare shown in brackets. Gly is the only residue without a side chain andthus imparts flexibility to the chain. Pro has an unusual geometry whichgreatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. Techniques for introducing substitution mutations atpredetermined sites into DNA which has a known or partially knownsequence are well known and comprise M13 mutagenesis, for example. Themanipulation of DNA sequences for preparing proteins havingsubstitutions, insertions or deletions, is described in detail inSambrook et al. (1989), for example.

According to the invention, “derivatives” of proteins or polypeptidesalso comprise single or multiple substitutions, deletions and/oradditions of any molecules associated with the enzyme, such ascarbohydrates, lipids and/or proteins or polypeptides. The term“derivative” also extends to all functional chemical equivalents of saidproteins or polypeptides.

According to the invention, a part or fragment of a tumor-associatedantigen has a functional property of the polypeptide from which it hasbeen derived. Such functional properties comprise the interaction withantibodies, the interaction with other polypeptides or proteins, theselective binding of nucleic acids and an enzymatic activity. Aparticular property is the ability to form a complex with HLA and, whereappropriate, generate an immune response. This immune response may bebased on stimulating cytotoxic or T helper cells. A part or fragment ofa tumor-associated antigen of the invention preferably comprises asequence of at least 6, in particular at least 8, at least 10, at least12, at least 15, at least 20, at least 30 or at least 50, consecutiveamino acids of the tumor-associated antigen. A part or fragment of atumor-associated antigen is preferably a part of the tumor-associatedantigen which corresponds to the non-transmembrane portion, inparticular the extracellular portion of the antigen or is comprisedthereof.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the invention to the part of the nucleicacid, which codes at least for the tumor-associated antigen and/or for apart or a fragment of said tumor-associated antigen, as defined above.Preferably, a part or fragment of a nucleic acid coding for atumor-associated antigen is that part which corresponds to the openreading frame, in particular as indicated in the sequence listing.

The isolation and identification of genes coding for tumor-associatedantigens also make possible the diagnosis of a disease characterized byexpression of one or more tumor-associated antigens. These methodscomprise determining one or more nucleic acids which code for atumor-associated antigen and/or determining the encoded tumor-associatedantigens and/or peptides derived therefrom. The nucleic acids may bedetermined in the conventional manner, including by polymerase chainreaction or hybridization with a labeled probe. Tumor-associatedantigens or peptides derived therefrom may be determined by screeningpatient antisera with respect to recognizing the antigen and/or thepeptides. They may also be determined by screening T cells of thepatient for specificities for the corresponding tumor-associatedantigen.

The present invention also enables proteins binding to tumor-associatedantigens described herein to be isolated, including antibodies andcellular binding partners of said tumor-associated antigens.

According to the invention, particular embodiments ought to involveproviding “dominant negative” polypeptides derived from tumor-associatedantigens. A dominant negative polypeptide is an inactive protein variantwhich, by way of interacting with the cellular machinery, displaces anactive protein from its interaction with the cellular machinery or whichcompetes with the active protein, thereby reducing the effect of saidactive protein. For example, a dominant negative receptor which binds toa ligand but does not generate any signal as response to binding to theligand can reduce the biological effect of said ligand. Similarly, adominant negative catalytically inactive kinase which usually interactswith target proteins but does not phosphorylate said target proteins mayreduce phosphorylation of said target proteins as response to a cellularsignal. Similarly, a dominant negative transcription factor which bindsto a promoter site in the control region of a gene but does not increasetranscription of said gene may reduce the effect of a normaltranscription factor by occupying promoter binding sites, withoutincreasing transcription.

The result of expression of a dominant negative polypeptide in a cell isa reduction in the function of active proteins. The skilled worker mayprepare dominant negative variants of a protein, for example, byconventional mutagenesis methods and by evaluating the dominant negativeeffect of the variant polypeptide.

The invention also comprises substances such as polypeptides which bindto tumor-associated antigens. Such binding substances may be used, forexample, in screening assays for detecting tumor-associated antigens andcomplexes of tumor-associated antigens with their binding partners andin a purification of said tumor-associated antigens and of complexesthereof with their binding partners. Such substances may also be usedfor inhibiting the activity of tumor-associated antigens, for example bybinding to such antigens.

The invention therefore comprises binding substances such as, forexample, antibodies or antibody fragments, which are capable ofselectively binding to tumor-associated antigens. Antibodies comprisepolyclonal and monoclonal antibodies which are produced in theconventional manner.

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fcregions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

WO 92/04381 for example, describes production and use of humanizedmurine RSV antibodies in which at least part of the murine FR regionshave been replaced with FR regions of a human origin. Antibodies of thiskind, including fragments of intact antibodies with antigen-bindingcapability, are often referred to as “chimeric” antibodies.

The invention also provides F(ab′)₂, Fab, Fv, and Fd fragments ofantibodies, chimeric antibodies, in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, chimeric F(ab′)₂-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain-CDR3 regions have been replaced with homologous human or nonhumansequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, and chimeric Fd-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced with homologous human or nonhuman sequences. The invention alsocomprises “single-chain” antibodies.

Preferably, an antibody used according to the invention is directedagainst one of the sequences according to SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 61-68, 70, 72, 74, 76, 81, 82,86, 88, 96-101, 103, 105, 107, 109, 111, 113, or a part or derivativethereof and/or may be obtained by immunization using these peptides.

The invention also comprises polypeptides which bind specifically totumor-associated antigens. Polypeptide binding substances of this kindmay be provided, for example, by degenerate peptide libraries which maybe prepared simply in solution in an immobilized form or asphage-display libraries. It is likewise possible to preparecombinatorial libraries of peptides with one or more amino acids.Libraries of peptoids and nonpeptidic synthetic residues may also beprepared.

Phage display may be particularly effective in identifying bindingpeptides of the invention. In this connection, for example, a phagelibrary is prepared (using, for example, the M13, fd or lambda phages)which presents inserts of from 4 to about 80 amino acid residues inlength. Phages are then selected which carry inserts which bind to thetumor-associated antigen. This process may be repeated via two or morecycles of a reselection of phages binding to the tumor-associatedantigen. Repeated rounds result in a concentration of phages carryingparticular sequences. An analysis of DNA sequences may be carried out inorder to identify the sequences of the expressed polypeptides. Thesmallest linear portion of the sequence binding to the tumor-associatedantigen may be determined. The “two-hybrid system” of yeast may also beused for identifying polypeptides which bind to a tumor-associatedantigen. Tumor-associated antigens described according to the inventionor fragments thereof may be used for screening peptide libraries,including phage-display libraries, in order to identify and selectpeptide binding partners of the tumor-associated antigens. Suchmolecules may be used, for example, for screening assays, purificationprotocols, for interference with the function of the tumor-associatedantigen and for other purposes known to the skilled worker.

The antibodies described above and other binding molecules may be used,for example, for identifying tissue which expresses a tumor-associatedantigen. Antibodies may also be coupled to specific diagnosticsubstances for displaying cells and tissues expressing tumor-associatedantigens. They may also be coupled to therapeutically useful substances.Diagnostic substances comprise, in a nonlimiting manner, barium sulfate,iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate,meglumine diatrizoate, metrizamide, sodium tyropanoate and radiodiagnostic, including positron emitters such as fluorine-18 andcarbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131and indium-111, nuclides for nuclear magnetic resonance, such asfluorine and gadolinium. According to the invention, the term“therapeutically useful substance” means any therapeutic molecule which,as desired, is selectively guided to a cell which expresses one or moretumor-associated antigens, including anticancer agents, radioactiveiodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc.Anticancer agents comprise, for example, aminoglutethimide,azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine,dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil,interferon-C, lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Other anticancer agents are described, for example, in Goodmanand Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition,1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such aspokeweed antiviral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxinresidues may also be high energy-emitting radionuclides such ascobalt-60.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

According to the invention, the term “disease” refers to anypathological state in which tumor-associated antigens are expressed orabnormally expressed. “Abnormal expression” means according to theinvention that expression is altered, preferably increased, compared tothe state in a healthy individual. An increase in expression refers toan increase by at least 10%, in particular at least 20%, at least 50% orat least 100%. In one embodiment, the tumor-associated antigen isexpressed only in tissue of a diseased individual, while expression in ahealthy individual is repressed. One example of such a disease iscancer, in particular seminomas, melanomas, teratomas, gliomas, coloncancer, rectal cancer, kidney cancer, breast cancer, prostate cancer,cancer of the uterus, ovarian cancer, endometrial cancer, cancer of theesophagus, blood cancer, liver cancer, pancreatic cancer, skin cancer,brain cancer and lung cancer, lymphomas, and neuroblastomas. Examplesfor this are lung tumor, breast tumor, prostate tumor, colon tumor,renal cell carcinoma, cervical carcinoma, colon carcinoma and mammacarcinoma or metastases of the above cancer types or tumors.

According to the invention, a biological sample may be a tissue sampleand/or a cellular sample and may be obtained in the conventional mannersuch as by tissue biopsy, including punch biopsy, and by taking blood,bronchial aspirate, urine, feces or other body fluids, for use in thevarious methods described herein.

According to the invention, the term “immunoreactive cell” means a cellwhich can mature into an immune cell (such as B cell, T helper cell, orcytolytic T cell) with suitable stimulation. Immunoreactive cellscomprise CD34⁺ hematopoietic stem cells, immature and mature T cells andimmature and mature B cells. If production of cytolytic or T helpercells recognizing a tumor-associated antigen is desired, theimmunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic T lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-96/33265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor detecting specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).Soluble MHC class I molecules are folded in vitro in the presence of β₂microglobulin and a peptide antigen binding to said class I molecule.The MHC/peptide complexes are purified and then labeled with biotin.Tetramers are formed by mixing the biotinylated peptide-MHC complexeswith labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1.Tetramers are then contacted with cytotoxic T lymphocytes such asperipheral blood or lymph nodes. The tetramers bind to cytotoxic Tlymphocytes which recognize the peptide antigen/MHC class I complex.Cells which are bound to the tetramers may be sorted byfluorescence-controlled cell sorting to isolate reactive cytotoxic Tlymphocytes. The isolated cytotoxic T lymphocytes may then be propagatedin vitro.

In a therapeutic method referred to as adoptive transfer (Greenberg, J.Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynchet al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell59:603-614, 1989), cells presenting the desired complex (e.g. dendriticcells) are combined with cytotoxic T lymphocytes of the patient to betreated, resulting in a propagation of specific cytotoxic T lymphocytes.The propagated cytotoxic T lymphocytes are then administered to apatient having a cellular anomaly characterized by particular abnormalcells presenting the specific complex. The cytotoxic T lymphocytes thenlyse the abnormal cells, thereby achieving a desired therapeutic effect.

Often, of the T cell repertoire of a patient, only T cells with lowaffinity for a specific complex of this kind can be propagated, sincethose with high affinity have been extinguished due to development oftolerance. An alternative here may be a transfer of the T cell receptoritself. For this too, cells presenting the desired complex (e.g.dendritic cells) are combined with cytotoxic T lymphocytes of healthyindividuals. This results in propagation of specific cytotoxic Tlymphocytes with high affinity if the donor had no previous contact withthe specific complex. The high affinity T cell receptor of thesepropagated specific T lymphocytes is cloned and can be transduced viagene transfer, for example using retroviral vectors, into T cells ofother patients, as desired. Adoptive transfer is then carried out usingthese genetically altered T lymphocytes (Stanislawski et al., NatImmunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61, 2001).

The therapeutic aspects above start out from the fact that at least someof the abnormal cells of the patient present a complex of atumor-associated antigen and an HLA molecule. Such cells may beidentified in a manner known per se. As soon as cells presenting thecomplex have been identified, they may be combined with a sample fromthe patient, which contains cytotoxic T lymphocytes. If the cytotoxic Tlymphocytes lyse the cells presenting the complex, it can be assumedthat a tumor-associated antigen is presented.

Adoptive transfer is not the only form of therapy which can be appliedaccording to the invention. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting HLA molecule). Various cell types may be used. Furthermore,it is possible to use vectors which carry one or both of the genes ofinterest. Particular preference is given to viral or bacterial vectors.For example, nucleic acids coding for a tumor-associated antigen or fora part thereof may be functionally linked to promoter and enhancersequences which control expression of said tumor-associated antigen or afragment thereof in particular tissues or cell types. The nucleic acidmay be incorporated into an expression vector. Expression vectors may benonmodified extrachromosomal nucleic acids, plasmids or viral genomesinto which exogenous nucleic acids may be inserted. Nucleic acids codingfor a tumor-associated antigen may also be inserted into a retroviralgenome, thereby enabling the nucleic acid to be integrated into thegenome of the target tissue or target cell. In these systems, amicroorganism such as vaccinia virus, pox virus, Herpes simplex virus,retrovirus or adenovirus carries the gene of interest and de facto“infects” host cells. Another preferred form is the introduction of thetumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen-presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the HLA molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toHLA molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp et al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyet al., Proc Natl Acad Sci USA 98:3299-303, 2001). It may also becarried out in combination with reagents which facilitate uptake intodendritic cells. Preferred tumor-associated antigens comprise thosewhich react with allogenic cancer antisera or with T cells of manycancer patients. Of particular interest, however, are those againstwhich no spontaneous immune responses pre-exist. Evidently, it ispossible to induce against these immune responses which can lyse tumors(Keogh et al., J. Immunol. 167:787-96, 2001; Appella et al., Biomed PeptProteins Nucleic Acids 1:177-84, 1995; Wentworth et al., Mol Immunol.32:603-12, 1995).

The pharmaceutical compositions described according to the invention mayalso be used as vaccines for immunization. According to the invention,the terms “immunization” or “vaccination” mean an increase in oractivation of an immune response to an antigen. It is possible to useanimal models for testing an immunizing effect on cancer by using atumor-associated antigen or a nucleic acid coding therefor. For example,human cancer cells may be introduced into a mouse to generate a tumor,and one or more nucleic acids coding for tumor-associated antigens maybe administered. The effect on the cancer cells (for example reductionin tumor size) may be measured as a measure for the effectiveness of animmunization by the nucleic acid.

As part of the composition for an immunization, one or moretumor-associated antigens or stimulating fragments thereof areadministered together with one or more adjuvants for inducing an immuneresponse or for increasing an immune response. An adjuvant is asubstance which is incorporated into the antigen or administeredtogether with the latter and which enhances the immune response.Adjuvants may enhance the immune response by providing an antigenreservoir (extracellularly or in macrophages), activating macrophagesand stimulating particular lymphocytes. Adjuvants are known and comprisein a nonlimiting way monophosphoryl lipid A (MPL, SmithKline Beecham),saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham;WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells7:178-186, 1997), incomplete Freund's adjuvant, complete Freund'sadjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf. Krieg etal., Nature 374:546-9, 1995) and various water-in-oil emulsions preparedfrom biologically degradable oils such as squalene and/or tocopherol.Preferably, the peptides are administered in a mixture with DQS21/MPL.The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and in particular about 1:1. For administration tohumans, a vaccine formulation typically contains DQS21 and MPL in arange from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids. Examples of such costimulating molecules are B7-1 and B7-2 (CD80and CD86, respectively) which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cells. Thisinteraction provides a costimulation (signal 2) for anantigen/MHC/TCR-stimulated (signal 1) T cell, thereby enhancingpropagation of said T cell and the effector function. B7 also interactswith CTLA4 (CD152) on T cells, and studies involving CTLA4 and B7ligands demonstrate that B7-CTLA4 interaction can enhance antitumorimmunity and CTL propagation (Zheng, P. et al., Proc. Natl. Acad. Sci.USA 95(11):6284-6289 (1998)).

B7 is typically not expressed on tumor cells so that these are noeffective antigen-presenting cells (APCs) for T cells. Induction of B7expression would enable tumor cells to stimulate more effectivelypropagation of cytotoxic T lymphocytes and an effector function.Costimulation by a combination of B7/IL-6/IL-12 revealed induction ofIFN-gamma and Th1-cytokine profile in a T cell population, resulting infurther enhanced T cell activity (Gajewski et al., J. Immunol.154:5637-5648 (1995)).

A complete activation of cytotoxic T lymphocytes and a complete effectorfunction require an involvement of T helper cells via interactionbetween the CD40 ligand on said T helper cells and the CD40 moleculeexpressed by dendritic cells (Ridge et al., Nature 393:474 (1998),Bennett et al., Nature 393:478 (1998), Schönberger et al., Nature393:480 (1998)). The mechanism of this costimulating signal probablyrelates to the increase in B7 production and associated IL-6/IL-12production by said dendritic cells (antigen-presenting cells).CD40-CD40L interaction thus complements the interaction of signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28).

The use of anti-CD40 antibodies for stimulating dendritic cells would beexpected to directly enhance a response to tumor antigens which areusually outside the range of an inflammatory response or which arepresented by nonprofessional antigen-presenting cells (tumor cells). Inthese situations, T helper and B7-costimulating signals are notprovided. This mechanism could be used in connection with therapiesbased on antigen-pulsed dendritic cells or in situations in which Thelper epitopes have not been defined in known TRA precursors.

The invention also provides for administration of nucleic acids,polypeptides or peptides. Polypeptides and peptides may be administeredin a manner known per se. In one embodiment, nucleic acids areadministered by ex vivo methods, i.e. by removing cells from a patient,genetic modification of said cells in order to incorporate atumor-associated antigen and reintroduction of the altered cells intothe patient. This generally comprises introducing a functional copy of agene into the cells of a patient in vitro and reintroducing thegenetically altered cells into the patient. The functional copy of thegene is under the functional control of regulatory elements which allowthe gene to be expressed in the genetically altered cells. Transfectionand transduction methods are known to the skilled worker. The inventionalso provides for administering nucleic acids in vivo by using vectorssuch as viruses and target-controlled liposomes.

In a preferred embodiment, a viral vector for administering a nucleicacid coding for a tumor-associated antigen is selected from the groupconsisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e. they areincapable of generating infectious particles).

Various methods may be used in order to introduce according to theinvention nucleic acids into cells in vitro or in vivo. Methods of thiskind comprise transfection of nucleic acid CaPO₄ precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the invention may be administered inpharmaceutically compatible preparations. Such preparations may usuallycontain pharmaceutically compatible concentrations of salts, buffersubstances, preservatives, carriers, supplementing immunity-enhancingsubstances such as adjuvants (e.g. CpG oligonucleotides) and cytokinesand, where appropriate, other therapeutically active compounds.

The therapeutically active compounds of the invention may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the invention are administered in effective amounts.An “effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition characterized by expression of one or more tumor-associatedantigens, the desired reaction relates to inhibition of the course ofthe disease. This comprises slowing down the progress of the diseaseand, in particular, interrupting the progress of the disease. Thedesired reaction in a treatment of a disease or of a condition may alsobe delay of the onset or a prevention of the onset of said disease orsaid condition.

An effective amount of a composition of the invention will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors.

The pharmaceutical compositions of the invention are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The doses administered of the compositions of the invention may dependon various parameters such as the type of administration, the conditionof the patient, the desired period of administration, etc. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg, are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the invention are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition of the invention may comprise apharmaceutically compatible carrier. According to the invention, theterm “pharmaceutically compatible carrier” refers to one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the invention are usually such that no interaction occurs whichsubstantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the invention may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,parabens and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, solutions, suspensions, syrups, elixir or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

EXAMPLES Materials and Methods

The terms “in silico” and “electronic” refer solely to the utilizationof methods based on databases, which may also be used to simulatelaboratory experimental processes.

Unless expressly defined otherwise, all other terms and expressions areused so as to be understood by the skilled worker. The techniques andmethods mentioned are carried out in a manner known per se and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. All methods including the use of kitsand reagents are carried out according to the manufacturers'information.

A. Data Mining-Based Strategy for Identifying Tumor-Associated Antigens

According to the invention, public human protein and nucleic aciddatabases were screened with regard to cancer-specific antigensaccessible on the cell surface. The definition of the screening criteriarequired therefor, together with high throughput methods for analyzing,if possible, all proteins, formed the central component of thisstrategy.

The starting point consisted of the validated protein entries (NP) and,respectively, the corresponding mRNAs (NM) which have been deposited inthe RefSeq database (Pruitt et al., Trends Genet. January; 16(1):44-47,2000) of the National Center for Biotechnology Information (NCBI).Following the fundamental principle of gene→mRNA→protein, the proteinswere first studied for the presence of one or more transmembranedomains. To this end, the protein analysis program TMHMM server v.2.0(Krogh et al., Journal of Molecular Biology 305(3):567-580, 2001) wasused and the results thereof then verified again using the program ALOM2 (Nakai et al., Genomics 14:897-911, 1992). The prediction of furthersignal sequences which influenced the intracellular localisation ofproteins was done using the programs PSORT II (Horton et al.,Intelligent Systems for Molecular Biology 4:109-115, 1996) and iPSORT(Bannai et al., Bioinformatics, 18(2):298-305, 2002). The human NPfraction having a total of 19 110 entries provided 4634 filteredproteins.

The corresponding mRNA of each of these 4634 proteins, respectively, wasthen subjected to a homology search in the EST database (Boguski et al.,Nat. Genet. 4(4):332-333, 1993) of the NCBI with the aid of the BLASTalgorithm (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997). Thescreening criteria in this search were set to an e-value <10e-20 and aminimal sequence identity of 93% in such a way that the hits resultingtherefrom with high probability could only be derived from therespective mRNA but not from the homologous transcripts. Almost allmRNAs provided at least one hit in the EST database wherein in somecases the number of hits exceeded 4000.

Subsequently, the tissue-specific origin of the underlying cDNA libraryas well as the name of the library were determined for each of thesevalid hits. The tissues resulting therefrom were divided into 4different groups ranging from dispensable organs (group 3) to absolutelyessential organs (group 0). Another group, group 4, consisted of anysamples obtained from cancer tissue. The distribution of hits to thefive groups was recorded in a table which was sorted according to thebest ratio of the sum of groups 3 and 4 to the sum of groups 0-2. ThosemRNAs whose EST hits originated exclusively from cancer tissue reached atop position, followed by those which can additionally be found also intissues of dispensable organs of group 3. A further criterium for thesignificance of this distribution was the number of the independent cDNAlibraries from which the ESTs were obtained and was recorded in aseparate column of the table.

Since the transcripts determined in the first approach and thecorresponding proteins are firstly hypothetic constructs, furtherscreening criteria were used with the intention to prove the realexistence of the mRNAs and consequently also of the proteins. For thispurpose, each mRNA was compared to the predicted gene locus using theprogram “Spidey” (Wheelan et al., Genome Res. 11(11): 1952-1957, 2001).Only those transcripts which have at least one splicing process, i.e.which spread over at least 2 exons, were used for more detailedanalyses.

Sequential application of all the filters mentioned led to thetumor-associated antigens of the invention which can be consideredextracellularly accessible, owing to a predicted transmembrane domainand the topology related thereto. The expression profile derived fromthe EST data indicates, in all cases, cancer-specific expression whichmay at most extend only to dispensable organs.

B. Strategy of Validating the Tumor-Associated Antigens Identified by inSilico Analysis

In order to utilize the targets for immunotherapeutic purposes (antibodytherapy by means of monoclonal antibodies, vaccination, T-cellreceptor-mediated therapeutic approaches; cf. EP-B-0 879 282), in cancertherapy as well as for diagnostic problems, the validation of thetargets identified according to the invention is of central importance.In this connection, validation is carried out by expression analysis atboth RNA and protein levels.

1. Examination of RNA Expression

The identified tumor antigens are first validated with the aid of RNAwhich is obtained from various tissues or from tissue-specific celllines. Since the differential expression pattern of healthy tissue incomparison with tumor tissue is of decisive importance for thesubsequent therapeutic application, the target genes are preferablycharacterized with the aid of these tissue samples.

Total RNA is isolated from native tissue samples or from tumor celllines by standard methods of molecular biology. Said isolation may becarried out, for example, with the aid of the RNeasy Maxi kit (Qiagen,Cat. No. 75162) according to the manufacturer's instructions. Thisisolation method is based on the use of chaotropic reagent guanidiniumisothiocyanate. Alternatively, acidic phenol can be used for isolation(Chomczynski & Sacchi, Anal. Biochem. 162: 156-159, 1987). After thetissue has been worked up by means of guanidinium isothiocyanate, RNA isextracted with acidic phenol, subsequently precipitated with isopropanoland taken up in DEPC-treated water.

2-4 μg of the RNA isolated in this way are subsequently transcribed intocDNA, for example by means of Superscript II (Invitrogen) according tothe manufacturer's protocol. cDNA synthesis is primed with the aid ofrandom hexamers (e.g. Roche Diagnostics) according to standard protocolsof the relevant manufacturer. For quality control, the cDNAs areamplified over 30 cycles, using primers specific for the p53 gene whichis expressed only lowly. Only p53-positive cDNA samples will be used forthe subsequent reaction steps.

The antigens are analyzed in detail by carrying out an expressionanalysis by means of PCR or quantitative PCR (qPCR) on the basis of acDNA archive which has been isolated from various normal and tumortissues and from tumor cell lines. For this purpose, 0.5 μl of cDNA ofthe above reaction mixture is amplified by a DNA polymerase (e.g. 1 U ofHotStarTaq DNA polymerase, Qiagen) according to the protocols of theparticular manufacturer (total volume of the reaction mixture: 25-50μl). Aside from said polymerase, the amplification mixture comprises 0.3mM dNTPs, reaction buffer (final concentration 1×, depending on themanufacturer of the DNA polymerase) and in each case 0.3 mMgene-specific forward and reverse primers.

The specific primers of the target gene are, as far as possible,selected in such a way that they are located in two different exons sothat genomic contaminations do not lead to false-positive results. In anon-quantitative end point PCR, the cDNA is typically incubated at 95°C. for 15 minutes in order to denature the DNA and to activate theHot-Start enzyme. Subsequently the DNA is amplified over 35 cycles (1min at 95° C., 1 min at the primer-specific hybridization temperature(approx. 55-65° C.), 1 min at 72° C. to elongate the amplicons).Subsequently, 10 μl of the PCR mixture are applied to agarose gels andfractionated in the electric field. The DNA is made visible in the gelsby staining with ethidium bromide and the PCR result is documented byway of a photograph.

As an alternative to conventional PCR, expression of a target gene mayalso be analyzed by quantitative real time PCR. Meanwhile variousanalytical systems are available for this analysis, of which the bestknown ones are the ABI 7900 HT sequence detection system (AppliedBiosystems), the iCycler (Biorad) and the Light cycler (RocheDiagnostics). As described above, a specific PCR mixture is subjected toa run in the real time instruments. By adding a DNA-intercalating dye(e.g. ethidium bromide, CybrGreen), the newly synthesized DNA is madevisible by specific light excitation (according to the dyemanufacturers' information). A multiplicity of points measured duringamplification enables the entire process to be monitored and the nucleicacid concentration of the target gene to be determined quantitatively.The PCR mixture is normalized by measuring a housekeeping gene (e.g. 18SRNA, β-actin, GAPDH). Alternative strategies via fluorescently labeledDNA probes likewise allow quantitative determination of the target geneof a specific tissue sample (see TaqMan applications from AppliedBiosystems).

2. Cloning

The complete target gene which is required for further characterizationof the tumor antigen is cloned according to common molecular-biologicalmethods (e.g. in “Current Protocols in Molecular Biology”, John Wiley &Sons Ltd., Wiley InterScience). In order to clone the target gene or toanalyze its sequence, said gene is first amplified by a DNA polymerasehaving a proof reading function (e.g. pfu, Roche Diagnostics). Theamplicon is then ligated by standard methods into a cloning vector.Positive clones are identified by sequence analysis and subsequentlycharacterized with the aid of prediction programs and known algorithms.

3. Production of Antibodies

The tumor-associated antigens identified according to the invention arecharacterized, for example, by using antibodies. The invention furthercomprises the diagnostic or therapeutic use of antibodies. Antibodiesmay recognize proteins in the native and/or denatured state (Anderson etal., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J. Immunol. Methods234: 107-116, 2000; Kayyem et al., Eur. J. Biochem. 208: 1-8, 1992;Spiller et al., J. Immunol. Methods 224: 51-60, 1999).

Antisera comprising specific antibodies which specifically bind to thetarget protein may be prepared by various standard methods; cf., forexample, “Monoclonal Antibodies: A Practical Approach” by PhillipShepherd, Christopher Dean ISBN 0-19-963722-9, “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane ISBN: 0879693142 and “Using Antibodies:A Laboratory Manual: Portable Protocol NO” by Edward Harlow, David Lane,Ed Harlow ISBN: 0879695447. It is also possible here to generate affineand specific antibodies which recognize complex membrane proteins intheir native form (Azorsa et al., J. Immunol. Methods 229: 35-48, 1999;Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J.Immunol. Methods. 234: 107-116, 2000). This is especially important inthe preparation of antibodies which are intended to be usedtherapeutically but also for many diagnostic applications. For thispurpose, both the complete protein and extracellular partial sequencesmay be used for immunization.

Immunization and Production of Polyclonal Antibodies

Several immunization protocols have been published. A species (e.g.rabbits, mice) is immunized by a first injection of the desired targetprotein. The immune response of the animal to the immunogen can beenhanced by a second or third immunization within a defined period oftime (approx. 2-4 weeks after the previous immunization). Blood is takenfrom said animals and immune sera obtained, again after various definedtime intervals (1st bleeding after 4 weeks, then every 2-3 weeks, up to5 takings). The immune sera taken in this way comprise polyclonalantibodies which may be used to detect and characterize the targetprotein in Western blotting, by flow cytometry, immunofluorescence orimmunohistochemistry.

The animals are usually immunized by any of four well-establishedmethods, with other methods also in existence. The immunization may becarried out using peptides specific for the target protein, using thecomplete protein, using extracellular partial sequences of a proteinwhich can be identified experimentally or via prediction programs. Sincethe prediction programs do not always work perfectly, it is alsopossible to employ two domains separated from one another by atransmembrane domain. In this case, one of the two domains has to beextracellular, which may then be proved experimentally (see below). Theimmunization is provided by various commercial service providers.

(1) In the first case, peptides (length: 8-12 amino acids) aresynthesized by in vitro methods (possibly carried out by a commercialservice), and said peptides are used for immunization. Normally 3immunizations are carried out (e.g. with a concentration of 5-100μg/immunization).

(2) Alternatively, immunization may be carried out using recombinantproteins. For this purpose, the cloned DNA of the target gene is clonedinto an expression vector and the target protein is synthesized, forexample, cell-free in vitro, in bacteria (e.g. E. coli), in yeast (e.g.S. pombe), in insect cells or in mammalian cells, according to theconditions of the particular manufacturer (e.g. Roche Diagnostics,Invitrogen, Clontech, Qiagen). It is also possible to synthesize thetarget protein with the aid of viral expression systems (e.g.baculovirus, vacciniavirus, adenovirus). After it has been synthesizedin one of said systems, the target protein is purified, normally byemploying chromatographic methods. In this context, it is also possibleto use for immunization proteins which have a molecular anchor as an aidfor purification (e.g. His tag, Qiagen; FLAG tag, Roche Diagnostics; GSTfusion proteins). A multiplicity of protocols can be found, for example,in “Current Protocols in Molecular Biology”, John Wiley & Sons Ltd.,Wiley InterScience. After the target protein has been purified, animmunization is carried out as described above.

(3) If a cell line is available which synthesizes the desired proteinendogenously, it is also possible to use this cell line directly forpreparing the specific antiserum. In this case, immunization is carriedout by 1-3 injections with in each case approx. 1-5×10⁷ cells.

(4) The immunization may also be carried out by injecting DNA (DNAimmunization). For this purpose, the target gene is first cloned into anexpression vector so that the target sequence is under the control of astrong eukaryotic promoter (e.g. CMV promoter). Subsequently, DNA (e.g.1-10 μg per injection) is transferred as immunogen using a gene gun intocapillary regions with a strong blood flow in an organism (e.g. mouse,rabbit). The transferred DNA is taken up by the animal's cells, thetarget gene is expressed, and the animal finally develops an immuneresponse to the target protein (Jung et al., Mol. Cells 12: 41-49, 2001;Kasinrerk et al., Hybrid Hybridomics 21: 287-293, 2002).

Production of Monoclonal Antibodies

Monoclonal antibodies are traditionally produced with the aid of thehybridoma technology (technical details: see “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142, “Using Antibodies: A Laboratory Manual: PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).A new method which is also used is the “SLAM” technology. Here, B cellsare isolated from whole blood and the cells are made monoclonal.Subsequently the supernatant of the isolated B cell is analyzed for itsantibody specificity. In contrast to the hybridoma technology, thevariable region of the antibody gene is then amplified by single-cellPCR and cloned into a suitable vector. In this manner production ofmonoclonal antibodies is accelerated (de Wildt et al., J. Immunol.Methods 207:61-67, 1997).

4. Validation of the Targets by Protein-Chemical Methods UsingAntibodies

The antibodies which can be produced as described above can be used tomake a number of important statements about the target protein.Specifically the following analyses of validating the target protein areuseful:

Specificity of the Antibody

Assays based on cell culture with subsequent Western blotting are mostsuitable for demonstrating the fact that an antibody binds specificallyonly to the desired target protein (various variations are described,for example, in “Current Protocols in Proteinchemistry”, John Wiley &Sons Ltd., Wiley InterScience). For the demonstration, cells aretransfected with a cDNA for the target protein, which is under thecontrol of a strong eukaryotic promoter (e.g. cytomegalovirus promoter;CMV). A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75: 441-7, 1997). As an alternative, it is also possible to usecell lines which express the target gene endogenously (detection viatarget gene-specific RT-PCR). As a control, in the ideal case,homologous genes are cotransfected in the experiment, in order to beable to demonstrate in the following Western blot the specificity of theanalyzed antibody.

In the subsequent Western blotting, cells from cell culture or tissuesamples which might contain the target protein are lysed in a 1%strength SDS solution, and the proteins are denatured in the process.The lysates are fractionated according to size by electrophoresis on8-15% strength denaturing polyacrylamide gels (contain 1% SDS) (SDSpolyacrylamide gel electrophoresis, SDS-PAGE). The proteins are thentransferred by one of a plurality of blotting methods (e.g. semi-dryelectroblot; Biorad) to a specific membrane (e.g. nitrocellulose,Schleicher & Schüll). The desired protein can be visualized on thismembrane. For this purpose, the membrane is first incubated with theantibody which recognizes the target protein (dilution approx.1:20-1:200, depending on the specificity of said antibody), for 60minutes. After a washing step, the membrane is incubated with a secondantibody which is coupled to a marker (e.g. enzymes such as peroxidaseor alkaline phosphatase) and which recognizes the first antibody. It isthen possible to make the target protein visible on the membrane in acolor or chemiluminescent reaction (e.g. ECL, Amersham Bioscience). Anantibody with a high specificity for the target protein should in theideal case only recognise the desired protein itself.

Localization of the Target Protein

Various methods are used to confirm the membrane localization,identified in the in silico approach, of the target protein. Animportant and well-established method using the antibodies describedabove is immunofluorescence (IF). For this purpose, cells of establishedcell lines which either synthesize the target protein (detection of theRNA by RT-PCR or of the protein by Western blotting) or else have beentransfected with plasmid DNA are utilized. A wide variety of methods(e.g. electroporation, liposome-based transfection, calcium phosphateprecipitation) are well established for transfection of cell lines withDNA (e.g. Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). Theplasmid transfected, in immunofluorescence, may encode the unmodifiedprotein or else couple different amino acid markers to the targetprotein. The principle markers are, for example, the fluorescent greenfluorescent protein (GFP) in various differentially fluorescent forms,short peptide sequences of 6-12 amino acids for which high-affinity andspecific antibodies are available, or the short amino acid sequenceCys-Cys-X-X-Cys-Cys which can bind via its cysteines specificfluorescent substances (Invitrogen). Cells which synthesize the targetprotein are fixed, for example, with paraformaldehyde or methanol. Thecells may then, if required, be permeabilized by incubation withdetergents (e.g. 0.2% Triton X-100). The cells are then incubated with aprimary antibody which is directed against the target protein or againstone of the coupled markers. After a washing step, the mixture isincubated with a second antibody coupled to a fluorescent marker (e.g.fluorescein, Texas Red, Dako), which binds to the first antibody. Thecells labeled in this way are then overlaid with glycerol and analyzedwith the aid of a fluorescence microscope according to themanufacturer's information. Specific fluorescence emissions are achievedin this case by specific excitation depending on the substancesemployed. The analysis usually permits reliable localization of thetarget protein, the antibody quality and the target protein beingconfirmed in double stainings with, in addition to the target protein,also the coupled amino acid markers or other marker proteins whoselocalization has already been described in the literature being stained.GFP and its derivatives represent a special case, being excitabledirectly and themselves fluorescing. The membrane permeability which maybe controlled through the use of detergents, in immunofluorescence,allows demonstration of whether an immunogenic epitope is located insideor outside the cell. The prediction of the selected proteins can thus besupported experimentally. An alternative possibility is to detectextracellular domains by means of flow cytometry. For this purpose,cells are fixed under non-permeabilizing conditions (e.g. with PBS/Naazide/2% FCS/5 mM EDTA) and analyzed in a flow cytometer in accordancewith the manufacturer's instructions. Only extracellular epitopes can berecognized by the antibody to be analyzed in this method. A differencefrom immunofluorescence is that it is possible to distinguish betweendead and living cells by using, for example, propidium iodide or Trypanblue, and thus avoid false-positive results.

Another important detection is by immunohistochemistry (IHC) on specifictissue samples. The aim of this method is to identify the localizationof a protein in a functionally intact tissue aggregate. IHC servesspecifically for (1) being able to estimate the amount of target proteinin tumor and normal tissues, (2) analyzing how many cells in tumor andhealthy tissues express the target gene, and (3) defining the cell typein a tissue (tumor, healthy cells) in which the target protein isdetectable. Alternatively, the amounts of protein of a target gene maybe quantified by tissue immunofluorescence using a digital camera andsuitable software (e.g. Tillvision, Till-photonics, Germany). Thetechnology has frequently been published, and details of staining andmicroscopy can therefore be found, for example, in “DiagnosticImmunohistochemistry” by David J., MD Dabbs ISBN: 0443065667 or in“Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: ForLight and Electron Microscopy” ISBN: 0306467704. It should be notedthat, owing to the properties of antibodies, different protocols have tobe used (an example is described below) in order to obtain a meaningfulresult.

Normally, histologically defined tumor tissues and, as reference,comparable healthy tissues are employed in IHC. It is also possible touse as positive and negative controls cell lines in which the presenceof the target gene is known through RT-PCR analyses. A backgroundcontrol must always be included.

Formalin-fixed (another fixation method, for example with methanol, isalso possible) and paraffin-embedded tissue pieces with a thickness of 4μm are applied to a glass support and deparaffinated with xylene, forexample. The samples are washed with TBS-T and blocked in serum. This isfollowed by incubation with the first antibody (dilution: 1:2 to 1:2000)for 1-18 hours, with affinity-purified antibodies normally being used. Awashing step is followed by incubation with a second antibody which iscoupled to an alkaline phosphatase (alternative: for example peroxidase)and directed against the first antibody, for approx. 30-60 minutes. Thisis followed by a color reaction using said alkaline phosphatase (cf.,for example, Shi et al., J. Histochem. Cytochem. 39: 741-748, 1991; Shinet al., Lab. Invest. 64: 693-702, 1991). To demonstrate antibodyspecificity, the reaction can be blocked by previous addition of theimmunogen.

Analysis of Protein Modifications

Secondary protein modifications such as, for example, N- andO-glycosylations or myristilations may impair or even completely preventthe accessibility of immunogenic epitopes and thus call into questionthe efficacy of antibody therapies. Moreover, it has frequently beendemonstrated that the type and amount of secondary modifications differin normal and tumor tissues (e.g. Durand & Seta, 2000; Clin. Chem. 46:795-805; Hakomori, 1996; Cancer Res. 56: 5309-18). The analysis of thesemodifications is therefore essential to the therapeutic success of anantibody. Potential binding sites can be predicted by specificalgorithms.

Analysis of protein modifications usually takes place by Westernblotting (see above). Glycosylations which usually have a size ofseveral kDa, especially lead to a larger total mass of the targetprotein, which can be fractionated in SDS-PAGE. To detect specific O-and N-glycosidic bonds, protein lysates are incubated prior todenaturation by SDS with O- or N-glycosylases (in accordance with theirrespective manufacturer's instructions, e.g. PNgase, endoglycosidase F,endoglycosidase H, Roche Diagnostics). This is followed by Westernblotting as described above. Thus, if there is a reduction in the sizeof a target protein after incubation with a glycosidase, it is possibleto detect a specific glycosylation and, in this way, also analyze thetumor specificity of a modification.

Functional Analysis of the Target Gene

The function of the target molecule may be crucial for its therapeuticusefulness, so that functional analyses are an important component inthe characterization of therapeutically utilizable molecules. Thefunctional analysis may take place either in cells, in cell cultureexperiments or else in vivo with the aid of animal models. This involveseither switching off the gene of the target molecule by mutation(knockout) or inserting the target sequence into the cell or theorganism (knockin). Thus it is possible to analyze functionalmodifications in a cellular context firstly by way of the loss offunction of the gene to be analyzed (loss of function). In the secondcase, modifications caused by addition of the analyzed gene can beanalyzed (gain of function).

A. Functional Analysis in Cells

Transfection. In order to analyze the gain of function, the gene of thetarget molecule must be transferred into the cell. For this purpose,cells are transfected with a DNA which allows synthesis of the targetmolecule. Normally, the gene of the target molecule here is under thecontrol of a strong eukaryotic promoter (e.g. cytomegalovirus promoter;CMV). A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75: 441-7, 1997). The gene may be synthesized either transiently,without genomic integration, or else stably, with genomic integrationafter selection with neomycin, for example.

RNA interference (siRNA). An inhibition of expression of the targetgene, which may induce a complete loss of function of the targetmolecule in cells, may be generated by the RNA interference (siRNA)technology in cells (Hannon, G J. 2002. RNA interference. Nature 418:244-51; Czauderna et al. 2003. Nucl. Acid Res. 31: 670-82). For thispurpose, cells are transfected with short, double-stranded RNA moleculesof approx. 20-25 nucleotides in length, which are specific for thetarget molecule. An enzymic process then results in degradation of thespecific RNA of the target gene and thus in an inhibition of thefunction of the target protein and consequently enables the target geneto be functionally analyzed.

Cell lines which have been modified by means of transfection or siRNAmay subsequently be analyzed in different ways. The most common examplesare listed below.

1. Proliferation

A multiplicity of methods for analyzing cell proliferation areestablished and are commercially supplied by various companies (e.g.Roche Diagnostics, Invitrogen; details of the assay methods aredescribed in the numerous application protocols). The number of cells incell culture experiments can be determined by simple counting or bycolorimetric assays which measure the metabolic activity of the cells(e.g. wst-1, Roche Diagnostics). Metabolic assay methods measure thenumber of cells in an experiment indirectly via enzymic markers. Cellproliferation may be measured directly by analyzing the rate of DNAsynthesis, for example by adding bromodeoxyuridine (BrdU), with theintegrated BrdU being detected colorimetrically via specific antibodies.

2. Apoptosis and Cytotoxicity

A large number of assay systems for detecting cellular apoptosis andcytotoxicity are available. A decisive characteristic is the specific,enzyme-dependent fragmentation of genomic DNA, which is irreversible andresults in any case in death of the cell. Methods for detecting thesespecific DNA fragments are commercially obtainable. An additional methodavailable is the TUNEL assay which can detect DNA single-strand breaksalso in tissue sections. Cytotoxicity is mainly detected via an alteredcell permeability which serves as marker of the vitality state of cells.This involves on the one hand the analysis of markers which cantypically be found intracellularly in the cell culture supernatant. Onthe other hand, it is also possible to analyze the absorbability of dyemarkers which are not absorbed by intact cells. The best-known examplesof dye markers are Trypan blue and propidium iodide, a commonintracellular marker is lactate dehydrogenase which can be detectedenzymatically in the supernatant. Different assay systems of variouscommercial suppliers (e.g. Roche Diagnostics, Invitrogen) are available.

3. Migration Assay

The ability of cells to migrate is analyzed in a specific migrationassay, preferably with the aid of a Boyden chamber (Corning Costar)(Cinamon G., Alon R. J. Immunol. Methods. 2003 February; 273(1-2):53-62;Stockton et al. 2001. Mol. Biol. Cell. 12: 1937-56). For this purpose,cells are cultured on a filter with a specific pore size. Cells whichcan migrate are capable of migrating through this filter into anotherculture vessel below. Subsequent microscopic analysis then permitsdetermination of a possibly altered migration behavior induced by thegain of function or loss of function of the target molecule.

B. Functional Analysis in Animal Models

A possible alternative of cell culture experiments for the analysis oftarget gene function are complicated in vivo experiments in animalmodels. Compared to the cell-based methods, these models have theadvantage of being able to detect faulty developments or diseases whichare detectable only in the context of the whole organism. A multiplicityof models for human disorders are available by now (Abate-Shen & Shen.2002. Trends in Genetics 51-5; Matsusue et al. 2003. J. Clin. Invest.111:737-47). Various animal models such as, for example, yeast,nematodes or zebra fish have since been characterized intensively.However, models which are preferred over other species are animal modelssuch as, for example, mice (Mus musculus) because they offer the bestpossibility of reproducing the biological processes in a human context.For mice, on the one hand transgenic methods which integrate new genesinto the mouse genome have been established in recent years (gain offunction; Jegstrup I. et al. 2003. Lab Anim. 2003 January; 37(1):1-9).On the other hand, other methodical approaches switch off genes in themouse genome and thus induce a loss of function of a desired gene(knockout models, loss of function; Zambrowicz BP & Sands AT. 2003. Nat.Rev. Drug Discov. 2003 January; 2(1):38-51; Niwa H. 2001. Cell Struct.Funct. 2001 June; 26(3):137-48); technical details have been publishedin large numbers.

After the mouse models have been generated, alterations induced by thetransgene or by the loss of function of a gene can be analyzed in thecontext of the whole organism (Balling R, 2001. Ann. Rev. Genomics Hum.Genet. 2:463-92). Thus it is possible to carry out, for example,behavior tests as well as to biochemically study established bloodparameters. Histological analyses, immunohistochemistry or electronmicroscopy enable alterations to be characterized at the cellular level.The specific expression pattern of a gene can be detected by in-situhybridization (Peters et al. 2003. Hum. Mol. Genet 12:2109-20).

Example 1: Identification of the Hypothetical Protein FLJ31461 asDiagnostic and Therapeutic Cancer Target

Using gene prediction programs, F1131461 (SEQ ID NO: 1) filed under thegene bank accession number NM_152454 was determined as putativefunctionally not previously characterised gene on chromosome 15(15q25.3). Two possible open reading frames result from the sequencedeposited with the gene bank. The first reading frame encodes a proteinwith a length of 136 amino acids. The gene product (SEQ ID NO: 2) whichwas deposited in the RefSeq data bank of the NCBI under numberNP_689667, accordingly has a calculated molecular weight of about 15kDa. The second reading frame encodes a protein with a length of 100amino acids (nucleotide sequence: SEQ ID NO: 69; amino acid sequence:SEQ ID NO: 70).

In sequence analyses of the gene FLJ31461 cloned by us, we weresurprised to find the insertion of a nucleotide in the coding region incomparison to the sequences deposited in the databases. This results ina shifting of the reading frame. Two completely new open reading frames,which cannot be derived from the sequences already deposited in sequencedatabases, are the result. Hereby the new reading frame (SEQ ID NO: 71)encodes a new hypothetical protein with a length of 96 amino acids (SEQID NO: 72). SEQ ID NO: 73 encodes a hypothetical protein with the lengthof 133 amino acids (SEQ ID NO: 74). Because we have to assume, that theoriginal depositions with the databases are incorrect, we have focussedfurther investigations on SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73and SEQ ID NO: 74.

In accordance with the invention, after the establishment ofF1131461-specific quantitative RT-PCR (primer with the SEQ ID NO: 31,32, 91, 92, 93, 94) the quantity of gene-specific transcripts wasinvestigated in healthy tissue and in carcinoma samples (FIG. 1). Withthe exception of the testis, FLJ31461 cannot be detected in any of thenormal tissues investigated by us (FIG. 1A). FLJ31461 is therefore withgreat probability a strongly gamete-specific gene-product. Surprisingly,we found during the analysis of tumors that FLJ31461 is switched on inmany tumor types, while it is below the detection limit in thecorresponding normal tissues (FIG. 1A-D). This does not only apply tovirtually all breast tumors investigated by us (FIG. 1C) and also aseries of lung tumors and nose-throat carcinomas, but also otherneoplasias with varying frequency (FIG. 1D).

FLJ31461 is therefore a highly specific molecular marker for tumortissues, which may be used diagnostically as well as therapeutically. Asa typical representative of the class of so-calledcancer/testis-antigens, which due to their selective tissue distributionserve as markers, this gene product can for example guarantee theprecise targeting of tumor cells without damage to the normal tissues.Cancer/testis-genes are regarded as attractive target structures fortargeted therapies and are already tested for specific immunotherapeuticapproaches in cancerous diseases in phase I/II studies (i.e. Scanlan MJ, Gure A O, Jungbluth A A, Old L J, Chen Y T. 2002. Immunol. Rev. 2002October; 188: 22-32).

In order to confirm these data on protein level, specific antibodies orimmune sera have been generated by immunisation of animals. The proteintopology was predicted by analysis of the transmembrane domains of SEQID NO: 72 and SEQ ID NO: 74 with bioinformatics tools (TMHMM, TMPRED).In this way for SEQ ID NO: 72 for example two transmembrane domains werepredicted; the N-terminus and C-terminus of the protein areextracellular.

In accordance with the invention, peptide epitopes were chosen forimmunisation, particularly extracellular peptide epitopes, which arespecific for both protein variants.

Amongst others, the following peptides were selected for immunization inorder to produce antibodies: SEQ ID NO: 61, 62, 96, 97.

By way of example the data for the antibody produced by immunisationusing SEQ ID NO: 96, are shown. The specific antibody may be used undervarious fixation conditions for immunofluorescence investigations. Incomparative staining of RT-PCR-positive as well as negative cell-lines,the respective protein is in well detectable quantity specific amongstothers in those breast carcinoma cell-lines that were typed positiveusing quantitative RT-PCR (FIG. 2). The endogenous protein in this casepresents membrane-localised.

Such antibodies are suitable for immunohistochemical staining of humantissue sections. To a large extent we were able to confirm the tissuedistribution found on transcript level. While we observed hardly anyreactivity of the antibody in normal tissue with the exception of testistissue (FIG. 3A), antibodies against FLJ31461 stain various human tumorpreparations, amongst these the tumors of breast and lung (FIG. 3B). Thestaining of the cells occurs accentuated at the membranes, whichindicates a localisation of the protein at the cell surface.Surprisingly, we found that particularly metastases of tumors (FIG. 3B)express this protein particularly frequently and in a high proportion ofcells.

These data indicate on one hand, that this gene found by us indeed doesform a protein, that this protein is highly specific for human tumorsand that it is present on the surface membrane of such tumor cells.Therefore this protein is accessible particularly for therapeuticantibodies. Likewise, our data prove, that specific antibodies againstthis protein may be produced. These antibodies bind selectively via themarker FLJ31461 to tumor cells.

In accordance with the invention such antibodies may be used fordiagnostic purposes for example immunohistology. In particular, suchantibodies may be used therapeutically. The produced antibodies can alsobe used directly for the production of chimeric or humanised recombinantantibodies. This can also be done directly with antibodies obtained fromrabbits (cf. J. Biol Chem. 2000 May 5; 275(18):13668-76 by Rader C,Ritter G, Nathan S, Elia M, Gout I, Jungbluth A A, Cohen L S, Welt S,Old L J, Barbas C F 3^(rd) “The rabbit antibody repertoire as a novelsource for the generation of therapeutic human antibodies”). In order toachieve this, lymphocytes were taken from immunised animals. FLJ31461 isalso a highly attractive target for immunotherapeutic procedures, suchas vaccines or the adoptive transfer of antigen-specific T-lymphocytes.

Example 2: Identification of DSG4 (Desmoglein 4) as Diagnostic andTherapeutic Cancer Target

Gene DSG4 (desmoglein 4; SEQ ID NO: 75) with its translation product(SEQ ID NO: 76) is a member of the desmosomal cadherin-family. The geneconsists of 16 exons and is located on chromosome 18 (18q12). Thederived amino acid sequence encodes a precursor protein with a length of1040 amino acids. The processed protein (N-terminally truncated by 49amino acids) has a length of 991 amino acids and without modifications amolecular weight of about 108 kDa. It must be assumed that DSG4 is aglycosylised type 1 cell surface protein, just like other desmogleins.DSG4 was able to be detected as constituent of desmosomes (Kljuic et al.2003. Cell 113: 249-260). Desmosomes are complex intercellularconnections, which provide epithelial tissues (such as the epidermis)with mechanical stability. Auto-antibodies against other members of thedesmoglein-family appear to contribute to the loss of cell-cell-contactsin the epidermis by binding to desmosomes and appear to contribute tothe skin disease Pemphigus vulgaris. It has been described that DSG4 isnot expressed in most healthy tissues. Significant expression has todate only been reported for salivary gland, testis, prostate and skin(Whittock, Bower 2003. J Invest Derm 120: 523-530). A connection withtumor diseases has not been discussed previously.

In accordance with the invention, the expression was investigated onhealthy tissues and tumors using DSG4-specific oligonucleotides. SeveralDSG4-specific primer pairs were used for RT-PCR-investigations inaccordance with the invention. These are: DSG4 primer pair SEQ ID NO:77, 78 (exon 10 and exon 12), DSG4-primer pair SEQ ID NO: 83, 84 (exon 1and exon 5), DSG4-primer pair SEQ ID NO: 89, 90 (exon 5 and exon 8) andDSG4-primer pair SEQ ID NO: 95, 78 (exon 8 and exon 12).

The investigation using all primer pairs confirmed that DSG4 is notexpressed in most normal tissues. Depending on the primer pair howeverdifferent expression patters were observed (FIG. 4B). With primer pairsSEQ ID NO: 95, 78 (exons 8-12) no expression was detected in normaltissue, with the exception of a very slight expression in prostate andskin. Surprisingly, DSG4 can be detected using this primer pair in aseries of tumors. These are in particular tumors of the stomach, as wellas carcinomas of the mouth, nose and throat area (FIG. 4A).

With primer pairs SEQ ID NO: 77, 78 (exons 10-12) even the expression inthe above mentioned normal tissues of prostate and skin was lesspronounced. Surprisingly, with this primer pair a more pronouncedexpression was observed in tumors (FIG. 4A). On one hand these tumorsare those, which were conspicuous in investigations using the firstprimer pair, such as tumors of the stomach and carcinomas of the mouth,nose and throat area, but also other types of cancer (FIG. 4B, C). Inparticular in all intestinal tumors we detected a significant and highexpression, which we were not able to detect using the first primerpair. The expression in the various tumors was manifold above that inthe highest expressing toxicity-relevant normal tissue (FIG. 4B).

On the basis of these investigations, it appears that apart from thefull-length transcript SEQ ID NO: 75 and the protein derived therefrom(SEQ ID NO: 76) also truncated variants of DSG4 exist, which lackregions before exon 9 (FIG. 5).

An extended analysis of the gene locus of DSG4 showed, that variousvariants of the molecule must be expected having a deletion before exon9 (FIG. 5). These are the transcripts SEQ ID NO: 85, 87, 108, 110 and112 and their altered protein products SEQ ID NO: 86, 88, 109, 111 and113. The full-length transcript may also be modified in the regionsbeyond exon 10 and lead to variant transcripts SEQ ID NO: 102, 104, 106and proteins SEQ ID NO: 103, 105, 107.

The variants truncated before exon 9 are even more tumor-selective thanthe full-length variant and can be found in additional tumor types, suchas the colon carcinoma, in which the full-length variant is notexpressed. Because the transmembrane domain is located in exon 12, theregion amplified by primers SEQ ID NO: 77, 78 is extracellular andtherefore should be accessible to antibodies. This truncatedextracellular region contains the DSG4-gene sections exons 10, 11 and12. Therefore transcripts containing exons 10, 11 and 12 (SEQ ID NO: 79)of DSG4, are particularly suitable as diagnostic and therapeutic cancertargets. These regions of DSG4 code for a domain (SEQ ID NO: 81), whichis extracellular. Therefore DSG4-polynucleotides, which comprise exons10, 11, 12 (SEQ ID NO: 75, 79, 80, 85, 87, 106, 112) and thepolypeptides they encode (SEQ ID NO: 76, 81, 82, 86, 88, 107, 113) areparticularly useful as target structure of monoclonal antibodies inaccordance with the invention.

Accordingly, we have immunised animals with epitopes from the region ofthe full-length molecule (SEQ ID NO: 75) and from the extracellular areaof the truncated molecule (SEQ ID NO: 81), respectively.

We were able to generate antibodies, which stain the DSG4 on the surfaceof cells transfected with DSG4. Specific antibodies are then able tospecifically detect this protein using immunofluorescence (FIG. 6A) andflow cytometry (FIG. 6B) at the surface.

The pronounced expression and high incidence of this molecule for thepresented tumor indications make this protein, and particularly itstruncated variant, a highly interesting diagnostic and therapeuticmarker in accordance with the invention. This also includes thedetection of disseminated tumor cells in the serum, bone marrow andurine, as well as the detection of metastases in other organs usingRT-PCR in accordance to the invention.

The extracellular domain of DSG4, particularly the part close to thecell membrane, may be utilised as target structure of monoclonalantibodies for therapy as well as immune diagnosis in accordance withthe invention.

Furthermore, DSG4 can be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). In accordance with the invention, this comprises also thedevelopment of so-called “small compounds”, which modulate thebiological activity of DSG4 and can be used for the therapy of tumors.

Example 3: Identification of DSG3 (Desmoglein3) as Diagnostic andTherapeutic Cancer Target

The gene DSG3 (desmoglein3; SEQ ID NO: 3) and its translation product(SEQ ID NO: 4) is a member of the desmosomal cadherin-family, which ispublished at the NCBI under accession number NM_001944 (nucleotidesequence) or NP_001935 (protein sequence). The gene consists of 15 exonsand is located on chromosome 18 (18q12.1-q12.2). The derived amino acidsequence encodes a protein with 999 amino acids and a hypothetical sizeof about 130 kDa. DSG3 is a glycosylated type 1 cell surface protein andis able to be detected in desmosomes (Silos et al. J. Biol. Chem. 271:17504-17511, 1996). Desmosomes are complex intracellular connectionsconnecting the keratin filaments of adjacent cells in order to provideepithelial tissues (such as for example the epidermis) with mechanicalstability. The desmosomal cadherines desmoglein and desmocollin arecalcium-dependent adhesion molecules. Auto-antibodies againstdesmoglein3 and the resulting loss of cell-cell-contacts in theepidermis are involved in the skin disease Pemphigus vulgaris (Amagai etal., 1991. Cell 67: 869-877). This was also proven in animal models(Koch et al, 1997. J Cell Biol 5: 1091-1102).

In accordance with the invention, after establishment of a DSG3-specificquantitative RT-PCR (primer pair SEQ ID NO: 33, 34) the quantity ofgene-specific transcripts was investigated in healthy tissues andcarcinoma samples (FIG. 7; methods: compare Materials and Methods,Section B.1.). Our investigations demonstrated a differentialdistribution of the expression in normal tissues. DSG3 transcripts arehardly found in normal tissues. The only normal tissues expressingsignificant transcript quantities are the esophagus, skin and thymus(FIG. 7a ). In all other analysed tissues, particularly brain, heart,liver, pancreas, PBMC, lung, mamma, ovary, kidney, spleen, colon,lymphatic node, uterus, bladder and prostate, transcription is low ornot detectable (FIG. 7A). Surprisingly, we have been able to prove asignificant, to date not described expression of DSG3 in some tumortypes.

In quantitative RT-PCR-analyses of tumors DSG3-specific transcripts wereproven amongst others in tumors of the nose-throat area (“head neckcancer”) in a quantity, which exceeded that of the highest expressingtoxicity-relevant tissue (FIG. 7B). But also other tumors, such ascarcinomas of the esophagus (FIG. 7C), express this protein.

We have stained sections of human tissues with DSG3-specific antibodiesand were able to confirm the tumor-selectivity observed in the PCR (FIG.8).

The pronounced expression and high incidence of this molecule in thedescribed tumor-indications make this protein a highly interestingdiagnostic and therapeutic marker in accordance with the invention. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in the serum, bone marrow and urine, as well as thedetection of metastases in other organs using RT-PCR.

The extracellular domain of the type I membrane protein desmoglein3 (SEQID NO: 4, amino acids 1-611) located on the N-terminus can be used inaccordance with the invention as target structure of monoclonalantibodies for therapy as well as immune diagnosis. Furthermore, inaccordance with the invention, DSG3 can be used as vaccine (RNA, DNA,protein, peptides) for the induction of tumor-specific immune responses(T-cell and B-cell mediated immune reactions). In accordance with theinvention this comprises also the development of so-called “smallcompounds”, which modulate the biological activity of DSG3 and can beused for the therapy of tumors.

Example 4: Identification of the Transporter SLC6A3 (Solute CarrierFamily 6) as Diagnostic and Therapeutic Cancer Target

The gene SLC6A3 (SEQ ID NO: 5) and its translation product (SEQ ID NO:6) is a member of the sodium-neurotransmitter symporter family(SNF-family) and is deposited under accession number NM_001044(nucleotide sequence) or NP_001035 (protein sequence). The gene consistsof 16 exons and is located on chromosome 5 (5p15.3). The SLC6A3-geneencodes a glycoprotein with a length of 620 amino acids. SLC6A3 is anintegral membrane protein with a total of 12 transmembrane domains,which as homo-oligomer represents part of an ion-transporter complex(Hastrup et al., 2003. J Biol Chem 278: 45045-48).

In accordance with the invention, after the establishment of aSLC6A3-specific quantitative RT-PCR (primer pair SEQ ID NO: 35, 36) thedistribution of SLC6A3-specific transcripts was investigated in healthytissue and carcinoma samples (FIG. 9; methods: compare Materials andMethods, Section B.1.). In most normal tissues SLC6A3 is only little ornot at all expressed, a moderate expression of SLC6A3 was found only inthymus, spleen, ovary, pancreas as well as kidney. A significant, about100-fold increased overexpression of SLC6A3 was detected in kidneycarcinomas (FIG. 9A). A detailed analysis of the various kidney tissuesusing quantitative (FIG. 9B) and conventional RT-PCR (FIG. 9C)demonstrated, that SLC6A3 was expressed in 7/12 kidney cell carcinomasand overexpressed in 5/12 samples in comparison to non-tumorigenicsamples. A significantly lower but detectable SLC6A3-specific expressionwas also demonstrated in some tumor tissues of other carcinomas.Particularly in some mamma carcinomas, ovarian carcinomas, bronchialcarcinomas and prostate carcinomas SLC6A3-specific transcripts weredetected (FIGS. 9D and 9E).

In accordance with the invention, the various extracellular domains ofSLC6A3 can be used as target structures of monoclonal therapeuticantibodies. The following sequence regions with respect to SEQ ID NO: 6are predicted as extracellular for SLC6A3 (based on an analysis usingthe software TMHMM2): amino acids 89-97, 164-237, 288-310, 369-397,470-478, 545-558. The peptides listed under SEQ ID NO: 63 and 64 wereused for the production of SLC6A3-specific antibodies.

Example 5: Identification of GRM8 as Diagnostic and Therapeutic CancerTarget

The gene GRM8/GluR8 or “metabotrophic glutamate receptor 8” (SEQ ID NO:7) and its translation product (SEQ ID NO: 8) belongs to the family ofglutamate receptors. The gene consists of 10 exons and is located onchromosome 7 (7q31.3-q32.1). The protein encoded by the GRM8 gene has alength of 908 amino acids, its calculated molecular weight is 102 kDa.Prediction programs predict 7 transmembrane domains. The proteinexhibits a high homology (67% to 70% similarity) with GluR4 and GluR7(Scherrer et al., 1996. Genomics 31: 230-233).

L-glutamate is an important neurotransmitter in the central nervoussystem and activates ionotrophic as well as metabotrophic glutamatereceptors. GRM8-specific transcripts were to date only detected in thebrain or glia-cells. However, to date no investigations comparingtranscript or protein on a quantitative level of a larger number oftissues have been reported (Wu et al., 1998. Brain Res. 53: 88-97).

In accordance with the invention, after establishment of a GRM8-specificquantitative RT-PCR (primer pair SEQ ID NO: 37, 38) the distribution ofGRM8-specific transcripts was investigated in healthy tissue andcarcinoma samples (FIG. 10; methods: compare Materials and Methods,Section B.1.). Our investigations demonstrated a differentialdistribution of the expression in various normal tissues. We also foundGRM8-transcripts selectively not only in the brain, but also in lesserquantities in the tissues of the stomach, intestinum, bladder, ovary,lung and pancreas. In most other normal tissues GRM8 is significantlyless expressed or not at all detectable. In some tumors we were able todetect a significant, not previously described expression of GRM8.Particularly carcinomas of the colon, cervix and kidney cells exhibiteda more than 10-fold overexpression in comparison to all other normaltissues and are also distinctly above the expression level of braintissue (FIGS. 10A and 10B).

In accordance with the invention, the extracellular domains of GRM8 canbe used as target structures of therapeutic monoclonal antibodies. Withrespect to SEQ ID NO: 8, the amino acids 1-582, 644-652, 717-743 and806-819 are extracellularly localised.

Example 6: Identification of Cadherin 17 (CDH17) as Diagnostic andTherapeutic Cancer Target

The gene CDH17 (SEQ ID NO: 9) and its translation product (SEQ ID NO:10) is a member of the cadherin-family. The gene consists of 18 exonsand is located on chromosome 8 (8q22.1). It encodes a type 1transmembrane protein with a length of 832 amino acids, which withoutsecondary modifications has a calculated molecular weight of 92.1 kDaand which has one transmembrane domain. Cadherin 17 was cloned asproton-dependent peptide transporter by Dantzig et al. (Science 264:430-433, 1994). The calcium-dependent glycoprotein cadherin 17 contains7 cadherin-domains in the extracellular region (Gessner et al., Ann N YAcad Sci.; 915:136-43, 2000). The intracellular domain does not exhibitany homology with other cadherins. Expression investigations wereavailable only sporadically and not in the form of quantitativelycomparative transcript or protein investigations of a larger number ofdifferent tissues.

In accordance with the invention, after the establishment of aCDH17-specific quantitative RT-PCR (primer pair SEQ ID NO: 39, 40) thedistribution of CDH17-specific transcripts was investigated in healthytissue as well as carcinoma samples (FIG. 11; methods: compare Materialsand Methods, Section B.1.). In most normal tissues CDH17 is not at alldetectable (FIG. 11A). We found significant transcript quantitiesselectively in stomach and intestinal tissues, far less expression inbladder, spleen, lymph nodes, thymus, prostate and esophagus.Surprisingly, we detected a distinct, not previously describedCDH17-specific expression in tumors. For CDH17 in intestinal tumors andat least 2-10-fold overexpression was measured in comparison to normaltissues. CDH17 is also strongly expressed in stomach and esophagustumors (FIGS. 11B and 11C).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein a highly interestingdiagnostic and therapeutic marker in accordance with the invention. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in serum, bone marrow and urine, as well as the detection ofmetastases in other organs using RT-PCR.

In accordance with the invention, the extracellular domain of CDH17 canbe used as target structure of monoclonal antibodies for therapy as wellas immune diagnosis. With respect to SEQ ID NO: 10, the amino acids1-785 are localised extracellularly (prediction occurred using thesoftware TMHMM2).

Furthermore, CDH17 can be used as vaccine (RNA, DNA, protein, peptides)for the induction of tumor-specific immune responses (T-cell and B-cellmediated immune reactions) in accordance with the invention. Thisincludes in accordance with the invention also the development ofso-called “small compounds”, which modulate the biological activity ofCDH17 and can be used for the therapy of tumors.

Example 7: Identification of ABCC4 as Diagnostic and Therapeutic CancerTarget

The gene ABCC4 (SEQ ID NO: 11) and its translation product (SEQ ID NO:12) encode an ABC transporter (ATP-binding-cassette). The gene consistsof 31 exons and is located on chromosome 13 (13q31). It encodes aprotein with a length of 1325 amino acids, which without modificationshas a calculated molecular weight of about 149 kDa. ABCC4 is an integralmembrane protein. The topology of ABCC4 is not yet clarified, predictionprograms predict 12-14 transmembrane domains. ABC-transporters transportvarious molecules through extra- and intracellular membranes. ABCC4 is amember of the so-called MRP-family, of multi-drug-resistance proteins.The specific function of ABCC4 is not yet clarified, however it appearsthat the transporter plays a role in the cellular detoxification, whichis made responsible for the chemotherapeutic resistance of many tumors.

The tissue distribution of this gene product over the various organs ofthe human body has not yet been investigated. In accordance with theinvention, after establishment of an ABCC4-specific quantitative RT-PCR(primer pair SEQ ID NO: 41, 42) specific transcripts were investigatedin healthy tissue and in carcinoma samples (FIG. 12; methods: compareMaterials and Methods, Section B.1.). Our comparative investigations onall normal tissues confirm the published ubiquitous expression of ABCC4.ABCC4 was detected in all tested normal tissues. Surprisingly, we found,however, that in a number of tumors an overexpression of the transcriptexceeding the expression for normal tissues was observed. In thisrespect, ABCC4 is found in 2-15-fold increased quantity in comparison toall analysed normal tissues for example in tumors of the kidney andprostate as well as bronchial tumors (FIG. 12).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein a highly interestingdiagnostic and therapeutic marker in accordance with the invention. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in serum, bone marrow and urine, as well as the detection ofmetastases in other organs with the aid of RT-PCR.

In accordance with the invention, the extracellular domains of ABCC4 canbe used as target structures of monoclonal antibodies for therapy aswell as immune diagnosis. The exact localisation of the extracellulardomains is still unknown. With respect to SEQ ID NO: 12, the softwareTMHMM2 predicts the amino acids 114-132, 230-232, 347-350, 730-768,879-946 and 999-1325 as extracellular.

Furthermore, ABCC4 may be used as vaccine (RNA, DNA, protein, peptides)in accordance with the invention for the induction of tumor-specificimmune responses (T-cell and B-cell mediated immune reactions). Thisincludes in accordance with the invention also the development ofso-called “small compounds”, which modulate the biological activity ofABCC4 and can be used for the therapy of tumors.

Example 8: Identification of VIL1 as Diagnostic and Therapeutic CancerTarget

The gene VIL1 or “Villin1” (SEQ ID NO: 13) and its translation product(SEQ ID NO: 14) are encoded by a gene consisting of 19 exons onchromosome 2 (2q35-q36). The gene encodes a protein with 826 aminoacids, which without modifications has a calculated molecular weight ofabout 92 kDa. Villin is the structural main component of microvilli incells of the gastro-intestinal and urogenital epithelia. It represents acalcium-regulated, actin-binding protein.

Pringault et al. (EMBO J. 5: 3119-3124, 1986) cloned villin1 and wereable to prove the existence of two transcripts (2.7 kb and 3.5 kb).These variants arise due to the use of alternative polyadenylationsignals in the last exon. VIL1-specific transcripts were previouslydescribed in a multitude of tissues such as brain, heart, lung,intestine, kidney and the liver. However, previously no comprehensivequantitatively comparative transcript or protein investigations on alarger number of tissues were carried out, which might have giveninformation regarding the usefulness of VIL1 for therapeutic purposes.

In accordance with the invention, after establishment of a VIL1-specificquantitative RT-PCR (primer pair SEQ ID NO: 43, 44) the distribution ofthe specific transcripts in healthy tissue and carcinoma samples wereinvestigated (FIG. 13; methods: compare Materials and Methods, SectionB.1.). Our comparative investigations regarding all normal tissuesdemonstrate a differential distribution of the VIL1-specific expression.In almost all normal tissues VIL1-specific transcripts are notdetectable (FIG. 13A). In particular our findings disprove thepreviously described expression in brain, heart, breast, ovary, lymphnodes, esophagus, skin, thymus, bladder and muscle. We only foundVIL1-transcripts in stomach and intestine and a lower expression inpancreas, liver and PBMCs.

Surprisingly, however, we detected a significant, but previously notdescribed VIL1-specific overexpression in tumors. For example incarcinomas of the colon and stomach a 5- to 10-fold overexpression wasobserved in comparison to all analysed normal tissues (FIGS. 13A and13B). A significant VIL1-specific expression is also detectable intumors of the pancreas, stomach and liver as well as bronchial tumors.

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in serum, bone marrow and urine, as well as the detection ofmetastases in other organs with the aid of RT-PCR.

In accordance with the invention, it can be used as vaccine (RNA, DNA,protein, peptides) for the induction of tumors-specific immune responses(T-cell and B-cell mediated immune reactions). In accordance with theinvention, this also includes the development of so-called “smallcompounds”, which modulate the biological activity of VIL1 and can beused for the therapy of tumors.

Example 9: Identification of MGC34032 as Diagnostic and TherapeuticCancer Target

The translation product (SEQ ID NO: 16) of gene MGC34032 (SEQ ID NO: 15)is a hypothetical protein with currently unknown function. The geneconsists of 28 exons and is located on chromosome 1 (1p31.1). The geneencodes a protein with a length of 719 amino acids which has acalculated molecular weight of about 79 kDa. Prediction programsconsistently predict 8 transmembrane domains. Homologies are not known,publications regarding MGC34032 do not exist.

In accordance with the invention, after establishment of aMGC34032-specific quantitative RT-PCR (primer pair SEQ ID NO: 45, 46)the distribution of specific transcripts was investigated in healthytissue and carcinoma samples (FIG. 14; methods: compare Materials andMethods, Section B.1.). We found MGC34032-transcripts in all testednormal tissues. The comparison of transcript quantities in normaltissues with those found in tumors, however, showed surprisingly, thatvarious tumor-types exhibited a significant, not previously described 5-to 10-fold overexpression of this gene product. These are particularlycarcinomas of the esophagus, colon, ovary, lung and kidney cells as wellas ear-nose-throat carcinomas (FIG. 14A-D).

In order to produce MGC34032-specific antibodies the peptides listedunder SEQ ID NO: 98 and 99 were used. These antibodies were able stainMGC34032 at the cell surface (FIG. 15A).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisalso includes in accordance with the invention the detection ofdisseminated tumor cells in serum, bone marrow and urine, as well as thedetection of metastases in other organs with the aid of RT-PCR.

The extracellular domains of MGC34032 may be used in accordance with theinvention as target structures of monoclonal antibodies for therapy aswell as immune diagnosis. With respect to SEQ ID NO: 16, the amino acids62-240, 288-323, 395-461 and 633-646 are extracellularly localised(prediction occurred with the aid of the TMHMM2-software).

Furthermore, MGC34032 may be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This includes in accordance with the invention also thedevelopment of so-called “small compounds”, which modulate thebiological activity of MGC34032 and may be used for the therapy oftumors.

Example 10: Identification of the Serine Protease PRSS7 (Enterokinase)as Diagnostic and Therapeutic Cancer Target

The gene PRSS7 (SEQ ID NO: 17) and its translation product (SEQ ID NO:18) belong to the family of serine proteases. The gene consists of 25exons and is located on chromosome 21 (21q21). The gene encodes aprotein with a length of 1019 amino acids, which is further processedafter translation. The active enzyme consists of 2 peptide chains,connected by a disulfide-bridge, which are derived from a commonprecursor molecule through proteolytic cleavage. The heavy chainconsists of 784 amino acids. The light chain consisting of 235 aminoacids exhibits a distinct homology to known serine proteases. Predictionprograms predict one transmembrane domain for PRSS7. PRSS7 isparticularly formed in the apical cells and enterocytes of the smallintestine and therefore aids in the initial activation of theproteolytic enzymes of the pancreas (such as trypsin, chymotrypsin andcarboxypeptidase) (Imamura and Kitamoto, Am J Phsyiol Gastrointest LiverPhysiol 285: G1235-G1241, 2003). To date this protein had not beenassociated with human tumors.

In accordance with the invention, after establishment of aPRSS7-specific quantitative RT-PCR (primer pair SEQ ID NO: 47, 48) thedistribution of specific transcripts was investigated in healthy tissueand carcinoma samples (FIG. 16; methods: compare Materials and Methods,Section B.1.). In most analysed tissues we were not able to detectPRSS7-specific expression at all or only to a very small extent (FIG.16A). Relevant expression was only found in the duodenum (FIG. 16B).

PRSS7 is expressed by various tumor types. In a part of the investigatedstomach carcinomas a distinct overexpression was detected in comparisonto normal stomach tissue (FIG. 16B). Furthermore, carcinomas of theesophagus, liver as well as pancreas expressed PRSS7, in part the genewas distinctly overexpressed in some tumor samples in comparison to thecorresponding normal tissues (FIGS. 16B and 16C).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisalso includes in accordance with the invention the detection ofdisseminated tumor cells in serum, bone marrow and urine as well as thedetection of metastases in other organs with the aid of RT-PCR.

We have stained cells transfected by PRSS7, as well as sections of humantissues with PRSS7-specific antibodies and were able to confirm thepredicted protein topology on the membrane (FIGS. 17A and 17B).

The extracellular part of PRSS7 can be used in accordance with theinvention as target structure of monoclonal antibodies for therapy aswell as immune diagnosis. With respect to SEQ ID NO: 18, the amino acidsstarting from amino acid residue 50 are extracellularly localised.Furthermore, in accordance with the invention, PRSS7 can be used asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This includes in accordance with the invention also thedevelopment of so-called “small compounds”, which modulate thebiological activity of PRSS7 and may be used in the therapy of tumors.

Example 11: Identification of CLCA2 as Diagnostic and Therapeutic CancerTarget

The gene CLCA2 or “calcium activated chloride channel 2” (SEQ ID NO: 19)belongs to the family of chloride ion transporters. The gene consists of14 exons and is located on chromosome 1 (1p31-p22). The gene encodes aprotein with a length of 943 amino acids, which has a calculatedmolecular weight of about 120 kDa. Experimentally, 5 transmembranedomains as well as a large, N-terminally localised extracellular domainwere detected. CLCA2 is an ion-transporter (Gruber, 1999. Am J Physiol276, C1261-C1270).

CLCA2-transcripts were previously described in the lung, trachea and themammary gland (Gruber, 1999. Am J Physiol 276, C1261-C1270), as well asin the tissues of testis, prostate and uterus (Agnel, 1999. FEBS Letters435, 295-301). Comparative investigations in a comprehensive collectionof tissues were not previously available.

In accordance with the invention, after establishment of aCLCA2-specific quantitative RT-PCR (primer pair SEQ ID NO: 49, 50) thedistribution of specific transcripts was investigated in almost allhealthy tissues of the human body and in tumor samples (FIG. 18;methods: compare Materials and Methods, Section B.1.). We found adifferential expression of CLCA2 in normal tissues. In most analysedtissues transcription is not detectable. Only in the esophagus, skin,pancreas, and significantly less in thymus, bladder, colon and prostatewere we able to detect expression. Surprisingly, we found in some tumortypes significant, not previously described expression of CLCA2. Inparticular tumors of the nose-throat area, as well as breast, esophagus,ovary and pancreas carcinomas as well as bronchial carcinomas exhibiteda CLCA2-specific expression increased by a factor of 10 to 1000 incomparison to the corresponding normal tissues (FIG. 18).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisincludes in accordance with the invention also the detection ofdisseminated tumor cells in serum, bone marrow and urine as well as thedetection of metastases in other organs with the aid of RT-PCR.

The two extracellular domains (with respect to SEQ ID NO: 20; aminoacids 1-235, 448-552 and 925-943) may be used in accordance with theinvention as target structures of monoclonal antibodies for therapy aswell as in immune diagnosis.

By immunization using CLCA2-specific peptides (SEQ ID NO: 100, SEQ IDNO: 101) antibodies could be produced staining CLCA2 on the cellsurface. Cells transfected by CLCA2 express this protein on the cellmembrane (FIG. 19A). The tumor selectivity could be confirmed inimmunofluorescence using the specific antibody (FIG. 19B).

Furthermore, CLCA2 may be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This includes in accordance with the invention also thedevelopment of so-called “small compounds”, which modulate thebiological activity of CLCA2 and may be used for the therapy of tumors.

Example 12: Identification of TM4SF4 (“Transmembrane 4 SuperfamilyMember 4”) as Diagnostic and Therapeutic Cancer Target

The gene TM4SF4 (SEQ ID NO: 21) and its translation product (SEQ ID NO:22) is a member of the tetraspanin family (Hemler, 2001. J Cell Biol155, 1103-07). The gene consists of 5 exons and is located on chromosome3 (3q25).

The gene encodes a protein with a length of 202 amino acids and acalculated molecular weight of about 21.5 kDa. Prediction programsconsistently predict 4 transmembrane domains for TM4SF4. The protein isN-glycosylated in the region of the second extracellular domain and islocated in the cell membrane. It is described that the degree ofN-glycosylation has an effect on the regulation of the cellproliferation and that it is inhibited with increasing glycosylation(Wice & Gordon, 1995. J Biol Chem 270, 21907-18). Tetraspanines formcomplexes with various members of the group of integrins. Thesehigh-molecular multi-complexes are ascribed a multitude of importantfunctions in the cell. For example, they fulfil functions in thecell-cell-adhesion and in intercellular contacts, in the signaltransduction and in cell motility (Bereditschevski, 2001. J Cell Sci114, 4143-51).

TM4SF4-transcripts are described in the periportal area of the liver aswell as in specific sections of the intestine, but were not previouslyanalysed in other tissues and in particular not in tumors (Wice &Gordon, 1995. J Biol Chem 270, 21907-18). In accordance with theinvention, after establishment of a TM4SF4-specific quantitative RT-PCR(primer pair SEQ ID NO: 51, 52) the distribution of specific transcriptsin healthy tissue and in carcinoma samples was investigated (FIG. 20;methods: compare Materials and Methods, Section B.1.). Ourinvestigations showed a differential distribution of the expression innormal tissues. TM4SF4-specific transcripts were mainly found in samplesof normal liver tissue. In several other normal tissues (amongst otherspancreas) we found a distinctly lower expression (at least 10-fold).Expression was not detectable in the brain, heart muscle, skeletalmuscles, skin, breast tissue, ovary, PBMC, spleen, lymph nodes andcervix. Contrary to the published prediction, that TM4SF4 isdown-regulated in tumor tissue (Wice & Gordon, 1995. J Biol Chem 270,21907-18), at least comparable TM4SF4-specific expression was shown invarious tumors; in part TM4SF4 was overexpressed in tumors (FIG. 20A).In a detailed expression analysis we were also able to prove contrary topublished data, that TM4SF4 is not suppressed in liver tumors (FIG.20B). In addition, the gene was overexpressed in 4/12 colon tumorsamples in comparison to normal colon tissue (FIG. 20C).

In order to produce TM4SF4-specific antibodies, the peptides listedunder SEQ ID NO: 65 and 66 were used. These antibodies were able torecognise the TM4SF4-protein in various sizes, which represent putativeglycosylation patters (FIG. 21A). Furthermore, the surface localisationof TM4SF4 could be confirmed with the aid of immunofluorescence (FIG.21B) and the tumor-selectivity observed in the PCR could be confirmedwith the aid of immunhistological staining of human tissues (FIG. 21C).

In summary, TM4SF4 can be characterised as a membrane protein, whoseexpression is limited to cell-subpopulations of a few selected normaltissues. TM4SF4 is particularly detectable in the periportal hepatocytesin the liver and in the apical membrane of the epithelia of thegastro-intestinal tract. In the case of apical protein localisation, theprotein is not accessible in normal cells to antibodies, because in theintestinal epithelium it faces the lumen and therefore is not connectedto the vascular system. In intestinal tumors, however, these molecules,which are not accessible in healthy tissue, are no longer compartmenteddue to uncontrolled proliferation and the neovascularisation of thetumor, and are therefore accessible for therapeutic antibodies.

The two extracellular domains of TM4SF4 therefore may be used inaccordance with the invention as target structures of monoclonalantibodies. With respect to SEQ ID NO: 22, the amino acids 23-45 and110-156 are located extracelluarly (prediction was performed using thesoftware TMHMM2). For the peptides with the SEQ ID NO: 65 and 66polyclonal antibodies were already successfully generated (Wice &Gordon, 1995. J Biol Chem 270:21907-18). For therapeutic approaches forthe development of tumor-specific antibodies the peptides SEQ ID NO: 67and SEQ ID NO: 68 are suitable, which each contain a conserved motive“NXS/T” for posttranslational N-glycosylations, whereby “X” representsany amino acid except proline.

Example 13: Identification of CLDN19 as Diagnostic and TherapeuticCancer Target

The gene CLDN19 or claudin19 (SEQ ID NO: 23) with its translationproduct (SEQ ID NO: 24) is a member of the claudin family.

The gene encodes a protein with a length of 224 amino acids which has acalculated molecular weight of about 21.5 kDa. Prediction programsconsistently predict for claudin19 the 4 transmembrane domainscharacteristic for the family of claudins. Claudin19 to date has notbeen functionally characterised in greater detail. Functions have beendescribed for other members of the claudin-family. Accordingly, claudinsplay an important role in cell-cell-adhesion and in intercellularcontacts. They are part of large molecule complexes and so form membranepores (“tight junctions”) for cell-cell-contacts.

In accordance with the invention, after establishment of aCLDN19-specific quantitative RT-PCR (primer pair SEQ ID NO: 53, 54) thedistribution of specific transcripts was investigated in healthy tissueand carcinoma samples (FIG. 22; methods: compare Materials and Methods,Section B.1.). Surprisingly, we found a differential distribution of theexpression in normal tissues. In the majority of normal tissues (inparticular in the brain, heart muscle, skeletal muscle, liver, pancreas,PBMCs, lung, breast tissue, ovary, spleen, colon, stomach, lymph nodes,esophagus, skin and prostate) CLDN19 is not detectable. Only in normaltissue of the bladder, thymus and testis we were able to detectCLDN19-transcripts. The comparative investigation of tumor tissuesshowed surprisingly that CLDN19 is expressed by various tumors. Theseare particularly carcinomas of kidney, stomach, liver and breast, whichin comparison to corresponding normal tissues exhibit an up to 10-foldoverexpression. CLDN19 has not previously been described in the contextof human tumors (FIG. 22A-22E).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Inaccordance with the invention, this includes the detection ofdisseminated tumor cells in serum, bone mark and urine, as well as thedetection of metastases in other organs with the aid of RT-PCR.

The two extracellular domains (amino acids 28-76 and 142-160 withrespect to SEQ ID NO: 24) of CLDN19 may be used in accordance with theinvention as target structures of monoclonal antibodies for the therapyand immune diagnosis.

Furthermore, CLDN19 may be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). In accordance with the invention, this includes thedevelopment of so-called “small compounds”, which modulate thebiological activity of CLDN19 and may be used for the therapy of tumors.

Example 14: Identification of ALPPL2 as Diagnostic and TherapeuticCancer Target

The gene ALPPL2 or “stem cell-specific alkaline phosphatase” or GCAP(SEQ ID NO: 25) encodes a protein (SEQ ID NO: 26) belonging to thefamily of alkaline phosphatases (AP). This consists of four veryhomologous members in total (homology: 90-98%). The gene codes for atranscript with a length of 2486 bp and consists of 11 exons. ALPPL2 islocated on chromosome 2 (2q37.1) in the vicinity of its closely relatedfamily members ALPP and ALPI.

The derived protein has a length of 532 amino acids and a calculatedmolecular weight of about 57.3 kDa. ALPPL2 is glycosylated and locatedin the plasma membrane as homodimer via a GPI-anchor. The exactphysiological function of the enzyme is not known. For osteosarcomas orPaget's disease the alkaline phosphatase enzyme activity is used astumor marker (Millán, 1995. Crit Rev Clin Lab Sci 32, 1-39). However,this determination is non-specific and independent from the actualunderlying molecule. It is not clear, which of the three above mentionedphosphatases or possibly even other currently not known phosphatasesresult in this activity.

ALPPL2 has been used previously only as diagnostic marker “in situ” forthe diagnosis of gamete tumors (Roelofs et al., 1999. J Pathol 189,236-244).

In accordance with the literature concerning a limited initial set oftissue types, ALPPL2 is expressed in testis and in the thymus as well asin some stem cell tumors (LeDu, 2002. J Biol Chem 277, 49808-49814). Inaccordance with the invention after establishment of an ALPPL2-specificquantitative RT-PCR (primer pair SEQ ID NO: 55, 56) the distribution ofthis gene product was investigated in healthy tissue and in carcinomasamples, whereby a comprehensive diversity of tissues was investigated,which amongst others also represented all body tissues (FIG. 23;methods: compare Materials and Methods B.1.). We detected no protein inmost normal tissues (particularly in the brain, heart muscle, skeletalmuscle, liver, pancreas, PBMCs, breast tissue, ovary, spleen, colon,stomach, lymph nodes, esophagus, skin and prostate). We demonstratedexpression in normal tissues of testis and lung, and very low levels inthe thymus and colon. The comparative investigation of tumors, however,surprisingly showed that ALPPL2 is expressed in significant quantitiesby various tumor types, particularly in carcinomas of the colon,stomach, pancreas, ovary and lung, but also in carcinomas of thenose-throat area (FIGS. 23A and 23B).

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in the serum, bone marrow and urine, as well as thedetection of metastases in other organs with the aid of RT-PCR.

The entire ALPPL2-protein (SEQ ID NO: 26) is extracellularly located andtherefore can be used in accordance with the invention as a targetstructure for developing monoclonal antibodies for therapy as well asimmune diagnosis.

Furthermore, ALPPL2 in accordance with the invention can be used asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This includes in accordance with the invention also thedevelopment of so-called “small compounds”, which modulate thebiological activity of ALPPL2 and can be used in the therapy of tumors.

Example 15: Identification of GPR64 as Diagnostic and Therapeutic CancerTarget

The gene GPR64 or “G-protein coupled receptor 64” (SEQ ID NO: 27) andits translation product (SEQ ID NO: 28) belongs to a large group of7-transmembrane receptors. The gene encodes a transcript with a lengthof 3045 bp and consists of 27 exons. GPR64 is located on the chromosome(Xp22). The gene encodes a protein with a length of 987 amino acidswhich has a calculated molecular weight of about 108 kDa. The N-terminalregion represents an extracellular domain, which is stronglyglycosylated. The exact physiological function of this protein is notknown.

GPR64 has been investigated to date in only a small number of normaltissues, amongst which only the tissue of the epididymis was found toexpress this gene (Osterhoff, 1997. DNA Cell Biol 16, 379-389). Inaccordance with the invention we have established a GPR64-specificRT-PCR (primer pair SEQ ID NO: 57, 58) and have investigated thedistribution of this gene product in a comprehensive collection ofhealthy tissues (FIG. 24; methods: compare Materials and Methods,Section B.1.). In many normal tissues GPR64 is not detectable at all,some exhibit a low expression. Surprisingly, the investigation of thisprotein in tumors exhibited an overexpression, which was many timeshigher than that of the relevant normal tissues. For example, we foundsignificant overexpression in almost half of the ovary carcinomas (FIG.24A to 24C).

The pronounced expression and high incidence of this molecule in thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in serum, bone marrow and urine, as well as the detection ofmetastases in other organs with the aid of RT-PCR.

The four extracellular domains of GPR64 may be used in accordance withthe invention as target structures of monoclonal antibodies for therapyas well as immune diagnosis. With respect to SEQ ID NO: 28, the aminoacids 1-625, 684-695, 754-784 and 854-856 are located extracellularly.

Furthermore, GPR64 can be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This also includes in accordance with the invention thedevelopment of so-called “small compounds”, which modulate thebiological activity of GPR64 and may be used for the therapy of tumors.

Example 16: Identification of the Sodium/Potassium/Chloride TransporterSLC12A1 (Solute Carrier Family 12) as Diagnostic and Therapeutic CancerTarget

The gene SLC12A1 (SEQ ID NO: 29) encodes a translation product (SEQ IDNO: 30) and belongs to the family ofsodium-potassium-chloride-co-transporters. The gene consists of 26 exonsand is located on chromosome 15 (15q15-q21.1). It encodes a protein witha length of 1099 amino acids which has a calculated molecular weightwithout secondary modifications of about 120 kDa. SLC12A1 is an integralmembrane protein with 10 transmembrane domains. SLC12A1 mediates thereabsorption of sodium chloride in the Henle-Schleife and is the targetpoint of many clinically relevant diuretic agents (Quaggin et al.,Mammalian Genome 6: 557-561, 1995). Correspondingly, this molecule isprincipally accessible as target structure for medicaments, in otherwords it is “drugable”.

In accordance with the invention, after establishment of aSLC12A1-specific quantitative RT-PCR (primer pair SEQ ID NO: 59, 60) thedistribution of specific transcripts in healthy tissue and in carcinomasamples was investigated (FIG. 25). We confirmed that in normal tissuesthe expression of SLC12A1 is first and foremost limited to normal kidneytissue, as has also been described in the literature. In all othernormal tissues SLC12A1-specific transcripts are detectable in only verysmall quantities or not all (FIG. 25A). Surprisingly, in the comparativeanalysis of tumors we found an expression of SLC12A1. Especially incarcinomas of the kidney, breast, ovary and prostate (FIG. 25A to 25C)we found unexpectedly an up to 1,000,000-fold over-expression incomparison to the corresponding normal tissues (FIG. 25B to 25D).Previously, SLC12A1 has not been described in the context of tumordiseases.

The pronounced expression and high incidence of this molecule for thedescribed tumor indications make this protein in accordance with theinvention a highly interesting diagnostic and therapeutic marker. Thisincludes in accordance with the invention the detection of disseminatedtumor cells in serum, bone marrow and urine, as well as the detection ofmetastases in other organs with the aid of RT-PCR. The extracellulardomains of SLC12A1 may be used in accordance with the invention astarget structures of monoclonal antibodies for therapy and also immunediagnosis. With respect to SEQ ID NO: 30, the amino acids 1-181,234-257, 319-327, 402-415, 562-564 and 630-1099 are locatedextracellularly.

Furthermore, SLC12A1 can be used in accordance with the invention asvaccine (RNA, DNA, protein, peptides) for the induction oftumor-specific immune responses (T-cell and B-cell mediated immunereactions). This includes in accordance with the invention also thedevelopment of so-called “small compounds”, which modulate thebiological activity of SLC12A1 and may be used for the therapy oftumors.

1-32. (canceled)
 33. A method of detecting a cancer cell characterizedby expressing a tumor-associated antigen, which method comprisesdetecting or quantifying in a biological sample isolated from a patienta nucleic acid which codes for the tumor-associated antigen; wherein thetumor-associated antigen has an amino acid sequence as shown in SEQ IDNO: 72; and the detecting or quantifying comprises: (i) contacting thebiological sample with an agent which binds specifically to the nucleicacid coding for the tumor-associated antigen, and (ii) detecting orquantifying the formation of a complex between the agent and the nucleicacid or a part thereof; wherein the detection of a complex is indicativefor the presence of a cancer cell. 34-97. (canceled)
 98. The method ofclaim 33, wherein the cancer cell is from a cancer tissue which isselected from the group consisting of breast, lung, ear, nose, throat,esophagus, ovary, cervix, skin, prostate, kidney, colon, stomachpancreas, liver, and uterus.
 99. The method of claim 33, wherein thebiological sample comprises body fluid, body tissue, or a combinationthereof.
 100. The method of claim 33, wherein the agent is labeled witha detectable marker.
 101. The method of claim 100, wherein thedetectable marker is a radioactive marker or an enzymic marker.
 102. Themethod of claim 33, wherein the nucleic acid or the part thereof isdetected or quantified using a polynucleotide probe which hybridizesspecifically to said nucleic acid or to said part thereof.
 103. Themethod of claim 102, wherein the polynucleotide probe comprises asequence of 6-50 contiguous nucleotides of the nucleic acid coding forthe tumor-associated antigen.
 104. The method of claim 33, wherein thenucleic acid or the part thereof is detected or quantified byselectively amplifying said nucleic acid or said part thereof.
 105. Themethod of claim 33, wherein the nucleic acid or the part thereof isdetected or quantified by using a polynucleotide probe comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, and SEQ IDNO:
 94. 106. The method of claim 33, wherein the nucleic acid or thepart thereof is detected or quantified by using a polynucleotide probecomprising a nucleotide sequence according to SEQ ID NO:
 91. 107. Themethod of claim 33, wherein the nucleic acid comprises a nucleic acidsequence according to SEQ ID NO: 71 of the sequence listing.